JP6936383B2 - Progesterone measurement kit, progesterone measurement method and progesterone measurement reagent - Google Patents

Description

The present invention relates to a progesterone measuring kit, a progesterone measuring method, and a progesterone measuring reagent.

The fluorescence detection method is widely used as a highly sensitive and easy measurement method for quantifying proteins, enzymes, inorganic compounds and the like. The fluorescence detection method detects the presence of a substance to be measured by detecting the fluorescence emitted when the sample is irradiated with the excitation light of the specific wavelength, which is considered to contain the substance to be measured that is excited by light of a specific wavelength and emits fluorescence. This is a method to confirm. When the substance to be measured is not a phosphor, for example, when a substance in which a substance specifically bound to the substance to be measured is labeled with a fluorescent dye is brought into contact with the sample and then irradiated with excitation light in the same manner as described above. The presence of the substance to be measured can be confirmed by detecting the fluorescence emitted from the substance.

In the fluorescence detection method as described above, in order to improve the sensitivity for detecting a substance to be measured that is present in a trace amount, a method that utilizes the effect of increasing the electric field by plasmon resonance is known. In this method, in order to generate plasmon resonance, a sensor chip having a metal layer provided in a predetermined region on the transparent support is prepared, and the metal layer forming surface of the support is applied to the interface between the support and the metal layer. From the opposite surface side, the excitation light is incident at a predetermined angle equal to or greater than the total reflection angle. Surface plasmons are generated in the metal layer by irradiation with such excitation light, and the signal / noise ratio (S / N ratio) is improved by enhancing the fluorescence by the electric field enhancing action due to the generation of the surface plasmons, and the measurement is highly sensitive. Is possible. The fluorescence detection method by surface plasmon excitation (hereinafter, also referred to as “SPF method”) provides about 10 times the signal intensity of the fluorescence detection method by epi-radiation excitation (also referred to as epi-fluorescence fluorescence method) and measures with high sensitivity. can do.

Patent Document 1 describes fluorescent fine particles produced by blending an initial donor dye having a preferable excitation peak and a final acceptor dye having a preferable emission peak into polymer fine particles. Patent Document 1 describes that a polyazaindacene dye is used as the above dye.

Non-Patent Document 1 describes the design and synthesis of a novel dystilyl BODIPY® (abbreviation for boron-dipyrromethene) dye, and the synthesized dystyryl BODIPY® dye is contained in a chloromethane solution. Absorption and emission spectra at the site have been analyzed.

Further, in the immunoassay method, there is a test sample that reacts not only with a positive test sample containing a test substance but also with a negative test sample that does not contain a test substance and becomes positive. The problem of false positives has traditionally been recognized. The cause of such false positives has not been clarified, but it is thought that one of the causes is that some factor contained in serum causes a non-specific reaction.

As a technique for suppressing non-specific reactions, Patent Document 2 describes non-specific immunization of sensitized particles of 0.3 to 2.0 μm in an immunological measurement method, particularly an immunological measurement method using aggregation. It is described that in order to block the reaction, ultrafine particles of 0.2 μm or less in which a substance having a reactivity with a causative substance of a non-specific immune reaction is bound are used. In Patent Document 3, in a method of detecting a test substance by an immunoagglutination reaction using sensitized particles of 0.4 μm or more, insoluble carrier particles of 0.01 μm to 0.5 μm are used as particles used for blocking. Is described. Further, in Patent Document 4, for the purpose of suppressing a non-specific reaction, a method of adding an antibody that does not immunologically react with a non-measurement substance or an antigen immobilized on particles smaller than the particles that specifically react with each other. Is described. Further, Patent Document 5 uses an immunological measurement method using an immunological measurement particle in which an antibody or antigen that immunologically reacts with a substance to be measured is supported on a carrier having an average particle size of 0.05 to 0.5 μm. This non-specific reaction inhibitor is composed of an insoluble carrier carrying an antibody or antigen that does not immunologically react with the substance to be measured in the presence of an organic solvent, and the average particle size of the insoluble carrier is the average particle size of the carrier. Non-specific reaction inhibitors characterized by being smaller than are described. Patent Document 6 describes that in an immunodetection method using fluorescence spectroscopy, particles having an outer diameter of 1 μm or less suppress the influence of a non-specific reaction.

Patent Document 7 describes a detection method for detecting the amount of a substance to be detected by using a sensor chip including a sensor unit having a laminated structure including a metal layer adjacent to the dielectric plate on one surface of the dielectric plate. There is. Specifically, by bringing the sample into contact with the sensor unit, a fluorescently labeled binding substance composed of a fluorescent label and a binding substance labeled with the fluorescent label in an amount corresponding to the amount of the substance to be detected contained in the sample is produced. , By coupling on the sensor part and irradiating the sensor part with excitation light, an enhanced photoelectric field is generated on the sensor part, and the enhanced photoelectric field excites the fluorescent label, and the light generated due to the excitation. The amount of the substance to be detected is detected based on the amount of the substance to be detected. In Patent Document 7, the fluorescent label includes a plurality of first fluorescent dye molecules, and a plurality of first fluorescent dye molecules composed of a translucent material that transmits fluorescence generated from the plurality of first fluorescent dye molecules. A fluorescent substance composed of the first particles to be used is used. Further, as a blocking agent for preventing the fluorescent label-binding substance from being adsorbed on the sensor unit due to the non-specific adsorptivity of the fluorescent label-binding substance to the sensor unit, the first fluorescent dye molecule is not contained and the binding substance is specific. A blocking substance having no binding property and having a non-specific adsorptivity equivalent to that of the fluorescent label binding substance is used.

Patent Document 8 discloses an immunoassay method for suppressing nonspecific adsorption using dry particles having a defined particle size.

Patent Document 9 describes the importance of measuring progesterone, which is a luteal hormone, in an early pregnancy test. Progesterone measurements can draw conclusions about the ovulation cycle, estrous cycle, pregnancy or ataxia in various mammalian species.

Japanese Patent No. 3442777 Japanese Unexamined Patent Publication No. 60-256057 Japanese Unexamined Patent Publication No. 2000-22196 Japanese Unexamined Patent Publication No. 11-337551 Japanese Unexamined Patent Publication No. 2007-127438 JP-A-2010-19553 JP-A-2010-112748 JP 2015-072249 Special Table No. 8-503547 Gazette

Oliver Galangau et al. , Org. Biomol. Chem. , 2010, 8, 4546-4553

As described above, the SPF method is known as a method capable of highly sensitive measurement by a simple measurement method, but it is not sufficiently satisfactory for the measurement of a very small amount of a substance to be measured. rice field. Among the detection methods, in the competitive method for measuring small molecules such as progesterone that cannot be sandwiched by an antibody, the fluorescent label concentration in the reaction system is adjusted in order to increase the detection sensitivity in the region where the concentration of the substance to be measured is low. It is necessary to keep it low, but in that case, the fluorescence intensity of the substance to be measured on the high concentration region side is insufficient, and in the high concentration region, the error becomes very large, so that there is a problem that the substance to be measured cannot be measured accurately.

Further, as described in Patent Documents 2 to 6, there is a specific sample in which false positive due to a non-specific immune reaction causative substance present in the sample becomes a problem, and a substance that interacts with the non-specific immune reaction causative substance. It is disclosed that the problem of false positives is avoided by using the fine particles to which the above is added, but in the methods described in Patent Documents 2, 3, 4 and 5, the measurement by the aggregation method is low in sensitivity and very small amount. There was a problem that it could not be used to detect a substance to be measured. Further, the immunoassay method disclosed in Patent Document 2 has a drawback that it is not a simple measurement method because it requires a washing step and a centrifugation operation.

Patent Document 6 describes that in an immunodetection method using fluorescence spectroscopy, particles having an outer diameter of 1 μm or less suppress the influence of a non-specific reaction, but this is not a very simple measurement. Further, both Patent Documents 7 and 8 disclose a simple immunoassay method using a flow path and measure a sample as a measurement method using a fluorescence method, but in a region where the concentration of the substance to be measured is low. The technology for increasing the detection sensitivity is not disclosed.

By further increasing the sensitivity so that a trace amount of the detected substance can be detected, there is a problem that not only the problem of false positives due to non-specific adsorption but also noise increases.

The present invention prevents the problem of false positives due to non-specific adsorption in the measurement of progesterone in biological samples, suppresses the increase in generated noise, and measures progesterone with high accuracy in a wide range of concentrations from low to high concentrations. The problem to be solved is to provide a kit, a method and a reagent capable of realizing the above.

In order to solve the above-mentioned problems, the present inventors have prepared a labeled first particle modified with a first binding substance having a specific binding property to progesterone, and a specific binding property to the progesterone. For the second unlabeled particle modified with the second binding substance which does not have, the flow path for flowing the first particle and the second particle, and the first binding substance. The composition of the kit for measuring progesterone containing a substrate having a binding substance and containing the substance was intensively examined. As a result, as the labeled first particles, labeled particles having a maximum emission wavelength in a long wavelength region of 680 nm or more and showing a high quantum yield are used, and the average particle diameter of the first particles is 50 to 50 to. The above problem is solved by satisfying the conditions that the particle size is 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. I found out what I could do. The present invention has been completed based on these findings.

式中、Ｒ^１１〜Ｒ^１５はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよく、Ｒ^１１〜Ｒ^１５のうち少なくとも３つは水素原子以外の原子又は基を表す。Ｘ^１及びＸ^２はそれぞれ独立に、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、ヒドロキシ基、アルコキシ基、アリールオキシ基、アルキルチオ基、アリールチオ基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよく、Ｘ^１及びＸ^２は互いに連結して環を形成してもよい。Ａｒ^１及びＡｒ^２はそれぞれ独立に、アリール基又はヘテロ環基を表し、これらは置換基を有していてもよい。Ｌ^１及びＬ^２はそれぞれ独立に、式（Ｌ−１）〜式（Ｌ−４）の何れかを表す。

式中、Ｒ^１１１〜Ｒ^１１６はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。Ａは、−Ｏ−、−Ｓ−、又は−ＮＨ−を表す。That is, according to the present invention, the following invention is provided.
[1] A labeled first particle modified with a first binding substance having a specific binding property to progesterone,
An unlabeled second particle modified with a second binding agent that does not have specific binding to progesterone,
The flow path for flowing the first particle and the second particle, and
A substrate having a substance having a binding property to the first binding substance,
A progesterone measurement kit that contains
The first particle having the above-mentioned label contains at least one compound represented by the following formula (1) and the particle, and contains the particle.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. Large particle size,
kit.

In the formula, R ^{11 to} R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group. , Or arylthio groups, which may have substituents, and ^{at least three of R 11 to} R ¹⁵ represent atoms or groups other than hydrogen atoms. X ¹ and X ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group. It may have a substituent and X ¹ and X ² may be linked to each other to form a ring. Ar ¹ and Ar ² each independently represent an aryl group or a heterocyclic group, which may have a substituent. L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-.

[2] The kit according to [1], wherein the first particle and the second particle are latex particles.
[3] The first particle having the above-mentioned label is a luminescent particle containing at least one kind of energy donor compound, at least one kind of energy acceptor compound, and particles, and the above-mentioned energy donor compound and the above-mentioned energy. The kit according to [1] or [2], wherein at least one of the acceptor compounds is a compound represented by the above formula (1).
[4] The energy donor compound contains at least one compound represented by the above formula (1), and the energy acceptor compound contains at least one compound represented by the above formula (1) [3]. ] The kit described in.
[5] The kit according to [3] or [4], wherein the molar ratio of the energy donor compound to the energy acceptor compound is 1:10 to 10: 1.
[6] The kit according to any one of [1] to [5], wherein the mass ratio of the second particle to the first particle is 1 to 20.
[7] The kit according to any one of [1] to [6], wherein the first binding substance having specific binding property to progesterone is an antibody.

式中、Ｒ^１１〜Ｒ^１５はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよく、Ｒ^１１〜Ｒ^１５のうち少なくとも３つは水素原子以外の原子又は基を表す。Ｘ^１及びＸ^２はそれぞれ独立に、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、ヒドロキシ基、アルコキシ基、アリールオキシ基、アルキルチオ基、アリールチオ基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよく、Ｘ^１及びＸ^２は互いに連結して環を形成してもよい。Ａｒ^１及びＡｒ^２はそれぞれ独立に、アリール基又はヘテロ環基を表し、これらは置換基を有していてもよい。Ｌ^１及びＬ^２はそれぞれ独立に、式（Ｌ−１）〜式（Ｌ−４）の何れかを表す。

式中、Ｒ^１１１〜Ｒ^１１６はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。Ａは、−Ｏ−、−Ｓ−、又は−ＮＨ−を表す。[8] (i) (a) A labeled first particle modified with a first binding agent having a specific binding property to progesterone.
(B) An unlabeled second particle modified with a second binding agent that does not have specific binding to progesterone.
(C) A test sample containing progesterone and
To obtain a mixture by mixing
(Ii) A step of applying the mixture obtained in the above step (i) onto a substrate, and
(Iii) A step of capturing the first binding substance at a reaction site on the substrate having a substance having a binding property to the first binding substance.
(Iv) A step of detecting a labeled first particle modified with the first binding agent captured on the reaction site.
A method for measuring progesterone, including
The first particle having the above-mentioned label contains at least one compound represented by the following formula (1) and the particle, and contains the particle.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. Large particle size,
How to measure progesterone.

In the formula, R ^{11 to} R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group. , Or arylthio groups, which may have substituents, and ^{at least three of R 11 to} R ¹⁵ represent atoms or groups other than hydrogen atoms. X ¹ and X ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group. It may have a substituent and X ¹ and X ² may be linked to each other to form a ring. Ar ¹ and Ar ² each independently represent an aryl group or a heterocyclic group, which may have a substituent. L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-.

[9] The method according to [8], wherein the first particle and the second particle are latex particles.
[10] The first particle having the above-mentioned label is a luminescent particle containing at least one kind of energy donor compound, at least one kind of energy acceptor compound, and particles, and the above-mentioned energy donor compound and the above-mentioned energy. The method according to [8] or [9], wherein at least one of the acceptor compounds is a compound represented by the above formula (1).
[11] The energy donor compound contains at least one compound represented by the above formula (1), and the energy acceptor compound contains at least one compound represented by the above formula (1) [10]. ] The method described in.
[12] The method according to any one of [8] to [11], wherein the mass ratio of the second particle to the first particle is 1 to 20.
[13] In the step (iv), the first labeled particles modified with the first binding substance captured on the reaction site are detected by the surface plasmon fluorescence method, [8] to [12]. ] The method according to any one of.

式中、Ｒ^１１〜Ｒ^１５はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよく、Ｒ^１１〜Ｒ^１５のうち少なくとも３つは水素原子以外の原子又は基を表す。Ｘ^１及びＸ^２はそれぞれ独立に、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、ヒドロキシ基、アルコキシ基、アリールオキシ基、アルキルチオ基、アリールチオ基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよく、Ｘ^１及びＸ^２は互いに連結して環を形成してもよい。Ａｒ^１及びＡｒ^２はそれぞれ独立に、アリール基又はヘテロ環基を表し、これらは置換基を有していてもよい。Ｌ^１及びＬ^２はそれぞれ独立に、式（Ｌ−１）〜式（Ｌ−４）の何れかを表す。

式中、Ｒ^１１１〜Ｒ^１１６はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。Ａは、−Ｏ−、−Ｓ−、又は−ＮＨ−を表す。[14] (a) a labeled first particle modified with a first binding agent that has specific binding to progesterone, and (b) a second that does not have specific binding to progesterone. A progesterone-measuring reagent comprising a second unlabeled particle modified with a binding agent.
The first particle having the above-mentioned label contains at least one compound represented by the following formula (1) and the particle, and contains the particle.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. Large particle size,
Progesterone measurement reagent.

In the formula, R ^{11 to} R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group. , Or arylthio groups, which may have substituents, and ^{at least three of R 11 to} R ¹⁵ represent atoms or groups other than hydrogen atoms. X ¹ and X ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group. It may have a substituent and X ¹ and X ² may be linked to each other to form a ring. Ar ¹ and Ar ² each independently represent an aryl group or a heterocyclic group, which may have a substituent. L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-.

式中、Ｒ^１１、Ｒ^１２、Ｒ^１４、Ｒ^１５、Ｘ^１、Ｘ^２、Ａｒ^１、Ａｒ^２、Ｌ^１及びＬ^２は、式（１）における定義と同義であり、但し、Ｒ^１１、Ｒ^１２、Ｒ^１４及びＲ^１５の少なくとも２つは水素原子以外の原子又は基である。Ｒ^３１〜Ｒ^３５はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよく、Ｒ^３１、Ｒ^３２、Ｒ^３４及びＲ^３５のいずれか一つは２原子以上からなる基である。[15] The progesterone measuring reagent according to [14], wherein the compound represented by the above formula (1) is a compound represented by the following formula (3).

In the formula, R ¹¹ , R ¹² , R ¹⁴ , R ¹⁵ , X ¹ , X ² , Ar ¹ , Ar ² , L ¹ and L ² are synonymous with the definitions in formula (1), except that R ¹¹ , At ^{least two of R 12} , R ¹⁴ and R ¹⁵ are atoms or groups other than hydrogen atoms. R ^{31 to} R ³⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio. Representing a group, these may have a substituent ^{, and any one of R 31} , R ³² , R ³⁴ and R ³⁵ is a group consisting of two or more atoms.

式中、Ｒ^１２、Ｒ^１３、Ｒ^１４、Ｘ^１、Ｘ^２、Ａｒ^１、Ａｒ^２、Ｌ^１及びＬ^２は、式（１）における定義と同義であり、但し、Ｒ^１２、Ｒ^１３及びＲ^１４の少なくとも１つは水素原子以外の原子又は基である。Ｒ^４１及びＲ^４２はそれぞれ独立に、アリール基、ヘテロ環基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよい。[16] The progesterone measuring reagent according to [14], wherein the compound represented by the above formula (1) is a compound represented by the following formula (4).

In the formula, R ¹² , R ¹³ , R ¹⁴ , X ¹ , X ² , Ar ¹ , Ar ² , L ¹ and L ² are synonymous with the definitions in formula (1), except that R ¹² , R ¹³ and At ^{least one of R 14} is an atom or group other than a hydrogen atom. R ⁴¹ and R ⁴² each independently represent an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, which may have a substituent.

式中、Ｒ^１１〜Ｒ^１５、Ｘ^１、Ｘ^２、Ｌ^１及びＬ^２は、式（１）における定義と同義である。Ｒ^５１及びＲ^５２はそれぞれ独立に、アルキル基、アリール基、ヘテロアリール基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。Ｑ^１及びＱ^２はそれぞれ独立に、芳香族炭化水素環又は芳香族ヘテロ環を表し、これらは置換基を有していてもよい。[17] The progesterone measuring reagent according to [14], wherein the compound represented by the above formula (1) is a compound represented by the following formula (5).

In the formula, R ^{11 to} R ¹⁵ , X ¹ , X ² , L ¹ and L ² are synonymous with the definition in the formula (1). R ⁵¹ and R ⁵² each independently represent an alkyl group, an aryl group, a heteroaryl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group, and these have a substituent. You may. Q ¹ and Q ² each independently represent an aromatic hydrocarbon ring or aromatic hetero ring, which may have a substituent.

[18] The reagent for measuring progesterone according to any one of [14] to [17], wherein the mass ratio of the second particle to the first particle is 1 to 20.
[19] The reagent for measuring progesterone according to any one of [14] to [18], wherein the first binding substance having specific binding property to the progesterone is an antibody.
[20] The reagent for measuring progesterone according to any one of [14] to [19], wherein the first particle having the label is a fluorescent latex particle and the second particle is a latex particle.

According to the progesterone measurement kit, the progesterone measurement method, and the progesterone measurement reagent of the present invention, the problem of false positives due to nonspecific adsorption is prevented, the increase in generated noise is suppressed, and in a wide range of concentrations from low to high concentrations. Highly accurate measurement of progesterone can be realized.

FIG. 1 shows a schematic view of a sensor chip. FIG. 2 shows an exploded view of the sensor chip.

Hereinafter, embodiments of the present invention will be described in detail.
The numerical range indicated by using "~" in the present specification means a range including the numerical values before and after "~" as the minimum value and the maximum value, respectively.

[Progesterone measurement kit]
The progesterone measurement kit according to the present invention
A labeled first particle modified with a first binding agent that has specific binding to progesterone,
An unlabeled second particle modified with a second binding agent that does not have specific binding to progesterone,
The flow path for flowing the first particle and the second particle, and
A substrate having a substance having a binding property to the first binding substance,
A progesterone measurement kit that contains
The first particle having the above-mentioned label contains at least one compound and particles represented by the formula (1) specified in the present specification.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. Large particle size,
It is a kit.

(Biological sample)
In the present invention, progesterone in a biological sample can be measured.
The biological sample is not particularly limited as long as it is a sample that may contain progesterone. For example, a biological sample, particularly a body fluid of an animal (for example, human, dog, cat, horse, etc.) (for example, human, dog, cat, horse, etc.) For example, blood, serum, plasma, spinal fluid, tears, sweat, urine, pus, runny nose, or sputum) or excrement (eg, feces), organs, tissues, mucous membranes, skin, and the like can be mentioned.

(Progesterone)
Progesterone is a sex hormone secreted by the ovaries and placenta that is involved in luteal function and pregnancy. It is used to diagnose abnormal menstrual cycles and infertility. It is also used to confirm the mating time of dogs and the residual ovaries of cats.

(particle)
The material and form of the particles (first particles and second particles) in the present invention are not particularly limited, and for example, organic polymer particles such as polystyrene beads or inorganic particles such as glass beads can be used. Specific examples of particle materials include homopolymers obtained by polymerizing monomers such as styrene, methacrylic acid, glycidyl (meth) acrylate, butadiene, vinyl chloride, vinyl acetate acrylate, methyl methacrylate, ethyl methacrylate, phenyl methacrylate, or butyl methacrylate. , And a copolymer obtained by polymerizing two or more kinds of monomers, and the above homopolymer or a latex in which the copolymer is uniformly suspended may be used. Examples of the particles include other organic polymer powders, inorganic substance powders, microorganisms, blood cells, cell membrane fragments, liposomes, microcapsules and the like. Latex particles are preferable as the first particles and the second particles.

When latex particles are used, specific examples of the material of the latex include polystyrene, styrene-acrylic acid copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl (meth) acrylate copolymer, and styrene-styrene sulfonic acid. Examples thereof include salt copolymers, methacrylic acid polymers, acrylic acid polymers, acrylonitrile-butadiene-styrene copolymers, vinyl chloride-acrylic acid ester copolymers, and polyvinyl acetate acrylates. As the latex, a copolymer containing at least styrene as a monomer is preferable, and a copolymer of styrene and acrylic acid or methacrylic acid is particularly preferable. The method for producing the latex is not particularly limited, and the latex can be produced by any polymerization method. However, when the luminescent particles of the present invention are labeled with an antibody and used, the presence of a surfactant makes it difficult to immobilize the antibody. Therefore, in the production of latex, emulsifier-free emulsion polymerization, that is, a surfactant Emulsion polymerization that does not use an emulsifier such as is preferable.

(First binding substance)
The first binding substance used in the present invention is a substance having a specific binding property to progesterone. Antibodies can be used, but are not limited to, as the first binding agent. Preferably, the first binding agent is an antibody. When the first binding substance is an antibody, for example, an antiserum prepared from the serum of an animal immunized with the progesterone, an immunoglobulin fraction purified from the antiserum, or a spleen cell of an animal immunized with the progesterone. Monoclonal antibodies obtained by cell fusion using, or fragments thereof [eg, F (ab') ₂ , Fab, Fab', or Fv] and the like can be used. Preparation of these antibodies can be carried out by a conventional method. Further, the antibody may be modified as in the case of a chimeric antibody or the like, and a commercially available antibody or an antibody prepared by a known method from animal serum or culture supernatant can be used.

In the present invention, as the first binding substance, it is preferable to use an anti-progesterone antibody having specific binding property to progesterone (preferably, which specifically recognizes progesterone).

One of the methods for producing an anti-progesterone antibody will be described below as an example.
A progesterone-BSA conjugate can be prepared by mixing progesterone, bovine serum albumin (hereinafter abbreviated as BSA), and a condensing agent. The conjugate is used as a mouse immunosensitizing antigen and immunized several times subcutaneously on the back of the mouse. In this case, a complete adjuvant (CFA) and / or an incomplete adjuvant (Incomplete Fluid's Adjuvant: IFA) can be appropriately selected and used in combination with the immunosensitizing antigen. A complete adjuvant is a substance that stimulates immunity and is a mixture of paraffin and alasel. The incomplete adjuvant is one in which dead mycobacterium or tubercle bacillus is added to the complete adjuvant to further enhance the antigenicity. After performing immunosensitization several times in a few weeks, blood is collected from mice and antibody titers are measured. When a sufficient increase in antibody titer is observed, the antigen is administered intraperitoneally, and the spleen is removed several days later. By fusing the spleen cells thus removed from the immune mouse with the mutant myeloma cell (myeloma), a fused cell having an antibody-producing ability can be produced. Only antibody-producing cells against the target antigen are selected from these fusion cells, and limiting dilution is performed to proliferate only the cell line. The diluted cells can be cultured (cloned). By injecting the fusion cell line thus obtained into the abdominal cavity of a mouse and proliferating ascites-type antibody-producing cells, it becomes possible to produce a monoclonal antibody in the ascites, and these antibodies are recovered. By doing so, the target antibody can be obtained.

式（１）中の各記号の意味は、本明細書中に定義した通りである。(First particle with label)
The first particle having a label used in the present invention is a particle containing at least one compound represented by the following formula (1) and a particle, and is also referred to as a labeled particle or a fluorescently labeled particle. The first labeled particles are preferably latex particles, more preferably fluorescent latex particles.

The meaning of each symbol in the formula (1) is as defined in the present specification.

It is known that when the amount of ordinary dye compounds incorporated into particles is increased, the quantum yield is affected by the association (this is also called concentration quenching). In particular, a fluorescent dye compound having an absorption wavelength of 650 nm or more, which has a long wavelength, is easily quenched when incorporated into particles, and it is difficult to maintain a quantum yield.

The compound represented by the formula (1) used in the present invention is capable of long-wavelength emission by having a conjugated substituent, and has a plurality of substituents in the dipyrromethene skeleton in the polymer particles. The decrease in quantum yield can be suppressed. As a factor for suppressing the decrease in quantum yield, it is considered that the interaction between molecules (for example, π-π interaction) due to a plurality of substituents extending in the direction perpendicular to the dipyrromethene skeleton is suppressed. According to the compound represented by the formula (1), first particles (preferably fluorescent particles, more preferably fluorescent nanoparticles) having a label having high brightness can be produced particularly in a long wavelength region. When the first particle having a label is a fluorescent particle, the brightness means the fluorescence intensity. According to the present invention, since the emission quantum yield is high in the window region of the living body (around 650 to 900 nm, which is a near-infrared wavelength region that easily transmits the living body), it is possible to improve the sensitivity of sensing using light emission. be.

In the present specification, the alkyl group may be a linear group, a branched chain, a cyclic group, or a combination thereof, and the straight chain or branched chain alkyl group preferably has 1 to 36 carbon atoms, more preferably 1 to 36 carbon atoms. It is 18, more preferably 1 to 12, and particularly preferably 1 to 6. Examples of the cyclic alkyl group include cycloalkyl having 3 to 8 carbon atoms. Specific examples of the alkyl group include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, iso-butyl group, sec-butyl group, t-butyl group, n-pentyl group and n-hexyl. Group, n-heptyl group, n-octyl group, n-nonyl group, n-decyl group, n-undecyl group, n-dodecyl group, n-tridecyl group, n-tetradecyl group, n-pentadecyl group, n-hexadecyl group. Examples include a group, an n-heptadecyl group, an n-octadecyl group, a cyclohexyl group and the like.

In the present specification, the aryl group is preferably an aryl group having 6 to 48 carbon atoms, more preferably an aryl group having 6 to 24 carbon atoms, and further preferably an aryl group having 6 to 14 carbon atoms, for example. Examples thereof include a phenyl group, a naphthyl group, an aryl group, a pyrenyl group, a phenanthrenyl group, a biphenyl group and a fluorenyl group.

In the present specification, the heterocyclic group is preferably any of a 5- to 7-membered substituted or unsubstituted, saturated or unsaturated, aromatic or non-aromatic, monocyclic or condensed heterocyclic group. The heterocyclic group is preferably a heterocyclic group in which the ring-constituting atom is selected from a carbon atom, a nitrogen atom, an oxygen atom and a sulfur atom, and has at least one heteroatom of any one of the nitrogen atom, the oxygen atom and the sulfur atom. Yes, more preferably a 5- or 6-membered aromatic heterocyclic group having 3 to 30 carbon atoms. Examples of the heterocyclic group include a frill group, a benzofuryl group, a dibenzofuryl group, a thienyl group, a benzothienyl group, a dibenzothienyl group, a pyridyl group, a pyrimidinyl group, a quinolyl group, an isoquinolyl group, an acridinyl group and a phenanthridinyl group. Pteridinyl group, pyrazinyl group, quinoxalinyl group, pyrimidinyl group, quinazolyl group, pyridazinyl group, cinnolinyl group, phthalazinyl group, triazinyl group, oxazolyl group, benzoxazolyl group, thiazolyl group, benzothiazolyl group, imidazolyl group, benzoimidazolyl group, pyrazolyl group , Indazolyl group, isooxazolyl group, benzoisooxazolyl group, isothiazolyl group, benzoisothiazolyl group, oxadiazolyl group, thiadiazolyl group, triazolyl group, tetrazolyl group, furyl group, thienyl group, pyrrolyl group, indolyl group, imidazoly pyridini Examples thereof include a ru group and a carbazolyl group.

In the present specification, the acyl group is preferably a linear or branched alkanoyl group having 2 to 15 carbon atoms, and is, for example, an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, an isovaleryl group, or a pivaloyl group. , Hexanoyl group, heptanoyle group, benzoyl group and the like.

In the present specification, the alkoxy group is preferably an alkoxy group having 1 to 20 carbon atoms, for example, a methoxy group, an ethoxy group, a propoxy group, an n-butoxy group, a pentyloxy group, a hexyloxy group, and a heptyloxy group. The group and the like can be mentioned.
In the present specification, the aryloxy group is preferably an aryloxy group having 6 to 14 carbon atoms, and examples thereof include a phenoxy group, a naphthoxy group, and an anthryloxy group.

The alkylthio group is preferably an alkylthio group having 1 to 30 carbon atoms, and examples thereof include a methylthio group, an ethylthio group, an n-hexadecylthio group and the like.
The arylthio group is preferably an arylthio group having 6 to 30 carbon atoms, and examples thereof include a phenylthio group, a p-chlorophenylthio group, and an m-methoxyphenylthio group.

In the present specification, examples of the halogen atom include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

In the present specification, the aromatic ring refers to an aromatic hydrocarbon ring such as a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a pyrene ring, a perylene ring and a terylene ring; an inden ring, an azulene ring, a pyridine ring, a pyrazine ring, and the like. Pyrimidine ring, pyrazole ring, pyrazolidine ring, thiazolidine ring, oxazolidine ring, pyran ring, chromium ring, pyrrole ring, pyrrolidine ring, benzoimidazoline ring, imidazoline ring, imidazoline ring, imidazoline ring, pyrazole ring, triazole ring, triazine ring, Diazole ring, indolin ring, thiophene ring, thienothiophene ring, furan ring, oxazole ring, oxadiazole ring, thiazine ring, thiazole ring, indole ring, benzothiazole ring, benzothiazol ring, naphthozole ring, benzoxazole ring, naphtho Aromatic heterocycles such as oxazole ring, indorenin ring, benzoindrenin ring, pyrazine ring, quinoline ring and quinazoline ring; and condensed aromatic rings such as fluorene ring and carbazole ring, etc., which have 5 to 16 carbon atoms. Aromatic rings (condensed rings containing aromatic rings and aromatic rings) are preferable.
The aromatic ring may have a substituent, and the term "aromatic ring" means both an aromatic ring having a substituent and an aromatic ring having no substituent. Examples of the substituent contained in the aromatic ring include the substituents described in Substituent Group A described later.

As used herein, the amino group includes an amino group; a mono or dimethylamino group, a mono or diethylamino group and an alkyl substituted amino group such as a mono or di (n-propyl) amino group; a mono or diphenylamino group and a mono or diphenyl group. Amino groups substituted with aromatic residues such as naphthylamino groups; Amino groups substituted one by one with alkyl groups such as monoalkyl monophenylamino groups; benzylamino groups, acetylamino groups, phenylacetyl Amino groups and the like can be mentioned. Here, the aromatic residue means a group obtained by removing one hydrogen atom from the aromatic ring, and the aromatic ring is as described above in the present specification.

The alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio group represented by ^{R 11 to} R ^{15 has a substituent.} Examples of the substituent include the substituents described in the following substituent group A.

Substituent group A:
Sulfamoyl group, cyano group, isocyano group, thiocyanato group, isothiocyanato group, nitro group, nitrosyl group, halogen atom, hydroxy group, amino group, mercapto group, amide group, alkoxy group, aryloxy group, alkylthio group, arylthio group, carbamoyl Group, acyl group, aldehyde group, carbonyl group, aryl group, alkyl group, alkyl group substituted with halogen atom, ethenyl group, ethynyl group, silyl group, and trialkylsilyl group (trimethylsilyl group, etc.).

The alkyl group, aryl group, heterocyclic group, hydroxy group, alkoxy group, aryloxy group, alkylthio group, arylthio group, ethenyl group, or ethynyl group represented by ^{X 1} and X ^{2 may have a substituent.} As the above-mentioned substituent, the substituent described in the substituent group A can be mentioned.

The ^{aryl group or heterocyclic group represented by Ar 1} and Ar ² may have a substituent, and examples of the above-mentioned substituent include the substituents described in the substituent group A.
The alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio group represented by ^{R 111 to} R ^{116 has a substituent.} The substituent may include the substituent described in the substituent group A.

<Compound represented by formula (1)>
In the formula (1), R ^{11 to} R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group and aryloxy group. , Alkylthio group, or arylthio group, which may have a substituent. At ^{least three of R 11 to} R ¹⁵ represent atoms or groups other than hydrogen atoms, preferably ^{at least four of R 11 to} R 15 represent atoms or groups other than hydrogen atoms, and more preferably R ¹¹ to ^{R 15.} All of R ¹⁵ represent an atom or group other than a hydrogen atom.

R ¹¹ and R ¹⁵ may be the same or different atoms or groups, but are preferably the same atom or group. R ¹² and R ¹⁴ may be the same or different atoms or groups, but are preferably the same atom or group.
R ¹¹ and R ¹⁵ preferably represent a hydrogen atom, a halogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, which may have a substituent.
R ¹² and R ¹⁴ preferably represent alkyl groups, which may have substituents.
R ¹³ preferably represents an aryl group, which may have a substituent.

In formula (1), X ¹ and X ² are independently halogen atoms, alkyl groups, aryl groups, heterocyclic groups, hydroxy groups, alkoxy groups, aryloxy groups, alkylthio groups, arylthio groups, ethenyl groups, or ethynyl groups. Representing groups, they may have substituents and X ¹ and X ² may be linked to each other to form a ring.
X ¹ and X ² preferably represent a halogen atom or an alkoxy group. It is more preferable that X ¹ and X ² are a fluorine atom, a methoxy group, an ethoxy group, an isopropyloxy group, and a t-butyloxy group, and it is also preferable that these are substituted with a fluorine atom and an alkoxy group.

In formula (1), Ar ¹ and Ar ² each independently represent an aryl group or a heterocyclic group, which may have a substituent.

式中、Ｒ^１１１〜Ｒ^１１６はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。Ａは、−Ｏ−、−Ｓ−、又は−ＮＨ−を表す。
Ｌ^１及びＬ^２は、好ましくは、式（Ｌ−１）又は式（Ｌ−２）の何れかを表し、より好ましくは式（Ｌ−１）を表す。
Ｒ^１１１〜Ｒ^１１６は、好ましくは水素原子である。In the formula (1), L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-.
L ¹ and L ² preferably represent either the formula (L-1) or the formula (L-2), and more preferably the formula (L-1).
R ^{111 to} R ¹¹⁶ are preferably hydrogen atoms.

式中、Ｒ^１１〜Ｒ^１５、Ｘ^１、Ｘ^２、Ａｒ^１及びＡｒ^２は式（１）における定義と同義であり、好ましい範囲も、式（１）における好ましい範囲と同じである。Ｌ^２１及びＬ^２２はそれぞれ独立に、式（Ｌ−１）又は式（Ｌ−２）で表される基を表わす。Ｌ^２１及びＬ^２２は、好ましくは式（Ｌ−１）で表される基を表わす。

<About the compound represented by the formula (2)>
Preferred examples of the compound represented by the formula (1) include a compound represented by the following formula (2).

In the formula, R ^{11 to} R ¹⁵ , X ¹ , X ² , Ar ¹ and Ar ² are synonymous with the definition in the formula (1), and the preferable range is also the same as the preferable range in the formula (1). L ²¹ and L ²² each independently represent a group represented by the formula (L-1) or the formula (L-2). L ²¹ and L ²² preferably represent a group represented by the formula (L-1).

<About the compound represented by the formula (3)>
Preferred examples of the compound represented by the formula (1) include a compound represented by the following formula (3).

In formula (3), R ¹¹ , R ¹² , R ¹⁴ , R ¹⁵ , X ¹ , X ² , Ar ¹ , Ar ² , L ¹ and L ² are synonymous with the definitions in formula (1) and are in a preferable range. Is the same as the preferable range in the formula (1). However, ^{at least two of R 11} , R ¹² , R ¹⁴ and R ¹⁵ are atoms or groups other than hydrogen atoms, and preferably ^{at least three of R 11} , R ¹² , R ¹⁴ and R ¹⁵ are other than hydrogen atoms. Of, and more preferably, R ¹¹ , R ¹² , R ¹⁴ and R ¹⁵ are atoms or groups other than hydrogen atoms.

In formula (3), R ^{31 to} R ³⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, cyano group, acyl group, alkoxy group, respectively. It represents an aryloxy group, an alkylthio group, or an arylthio group, and these may have a substituent (the above-mentioned substituent includes the substituent described in the substituent group A), and R ³¹ and R ^32. , R ³⁴ and R ³⁵ are groups consisting of two or more atoms. As the group composed of two or more atoms, an alkyl group, an aryl group, an ethenyl group, an ethynyl group, an amino group, a cyano group and an alkoxy group are preferable, and an alkyl group is more preferable. Among the alkyl groups, an alkyl group composed of only a carbon atom and a hydrogen atom and an alkyl group substituted with a halogen atom are preferable, and an alkyl group and a fluorine atom composed of only carbon atoms having 1 to 6 carbon atoms and a hydrogen atom are preferable. Alkyl groups substituted with are more preferable, methyl groups, isopropyl groups, t-butyl groups and trifluoromethyl groups are even more preferable, and methyl groups are particularly preferable.

式（４）中、Ｒ^１２、Ｒ^１３、Ｒ^１４、Ｘ^１、Ｘ^２、Ａｒ^１、Ａｒ^２、Ｌ^１及びＬ^２は、式（１）における定義と同義であり、好ましい範囲も、式（１）における好ましい範囲と同じである。但し、Ｒ^１２、Ｒ^１３及びＲ^１４の少なくとも１つは水素原子以外の原子又は基であり、好ましくは、Ｒ^１２、Ｒ^１３及びＲ^１４の少なくとも２つは水素原子以外の原子又は基であり、より好ましくは、Ｒ^１２、Ｒ^１３及びＲ^１４は水素原子以外の原子又は基である。
式（４）中、Ｒ^４１及びＲ^４２はそれぞれ独立に、アリール基、ヘテロ環基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよい。上記置換基としては、置換基群Ａに記載の置換基が挙げられる。Ｒ^４１及びＲ^４２はそれぞれ独立に、アリール基、エテニル基、又はエチニル基であることが好ましく、量子収率向上の観点からは、アリール基が好ましく、長波長化の観点からは、エテニル基、エチニル基であることが好ましい。アリール基である場合、アリール基のオルト位又はメタ位に少なくとも一つ置換基を有していることが好ましく、オルト位に少なくとも一つ置換基を有していることがより好ましい。アリール基に置換する置換基の数は、１〜３つが好ましく、２つ又は３つがより好ましい。アリール基に置換する置換基としては、アルキル基であることが好ましく、メチル基、イソプロピル基、ｔ−ブチル基であることがより好ましく、メチル基であることがさらに好ましい。<About the compound represented by the formula (4)>
Preferred examples of the compound represented by the formula (1) include a compound represented by the following formula (4).

In the formula (4), R ¹² , R ¹³ , R ¹⁴ , X ¹ , X ² , Ar ¹ , Ar ² , L ¹ and L ² are synonymous with the definitions in the formula (1), and the preferable range is also the formula. It is the same as the preferable range in (1). However, ^{at least one of R 12} , R ¹³ and R ¹⁴ is an atom or group other than a hydrogen atom, and preferably ^{at least two of R 12} , R ¹³ and R ¹⁴ are atoms or groups other than a hydrogen atom. , More preferably, R ¹² , R ¹³ and R ¹⁴ are atoms or groups other than hydrogen atoms.
In formula (4), R ⁴¹ and R ⁴² each independently represent an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, which may have a substituent. Examples of the substituent include the substituents described in the substituent group A. Each of R ⁴¹ and R ⁴² is preferably an aryl group, an ethenyl group, or an ethynyl group independently, an aryl group is preferable from the viewpoint of improving the quantum yield, and an ethenyl group is preferable from the viewpoint of lengthening the wavelength. It is preferably an ethynyl group. In the case of an aryl group, it is preferable to have at least one substituent at the ortho-position or meta-position of the aryl group, and it is more preferable to have at least one substituent at the ortho-position. The number of substituents substituted with the aryl group is preferably 1 to 3, more preferably 2 or 3. The substituent to be substituted with the aryl group is preferably an alkyl group, more preferably a methyl group, an isopropyl group, or a t-butyl group, and even more preferably a methyl group.

<About the compound represented by the formula (5)>
Preferred examples of the compound represented by the formula (1) include a compound represented by the following formula (5).

In the formula (5), R ^{11 to} R ¹⁵ , X ¹ , X ² , L ¹ and L ² are synonymous with the definitions in the formula (1), and the preferable range is also the same as the preferable range in the formula (1). be.

In formula (5), R ⁵¹ and R ⁵² independently represent an alkyl group, an aryl group, a heteroaryl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group, respectively. It may have a substituent. Examples of the substituent include the substituents described in the substituent group A. R ⁵¹ and R ⁵² are preferably alkyl groups and alkoxy groups, respectively, and are more preferably alkyl groups from the viewpoint of improving quantum yield, and are methyl group, ethyl group, isopropyl group, and t-butyl. It is more preferably a group, and particularly preferably a methyl group. From the viewpoint of lengthening the wavelength, an alkoxy group is more preferable, a methoxy group, an ethoxy group, an isopropyloxy group, and a t-butyloxy group are more preferable, and a methoxy group is particularly preferable.

Q ¹ and Q ² each independently represent an aromatic hydrocarbon ring or aromatic hetero ring, which may have a substituent. Examples of the substituent include the substituents described in the substituent group A. Q ¹ and Q ² is preferably an aromatic hydrocarbon ring, a benzene ring, a naphthalene ring, anthracene ring, phenanthrene ring, more preferably a pyrene ring, a benzene ring, it is a naphthalene ring and more preferably , A benzene ring is particularly preferable. The group containing ^{R 51 and forming Q 1} and the group containing ^{R 52 and forming Q 2} are preferably a tolyl group, a xsilyl group and a mesityl group, more preferably a xsilyl group and a mesityl group, and L. ¹ or xylyl group having a methyl group at both ortho position with respect to the binding position with the L ^2, with a mesityl group having a methyl group at both ortho and para position with respect to the binding position with the L ¹ or L ² It is more preferable to have a mesityl group having a methyl group at both the ortho position and the para position with respect to the bond position with ^{L 1} or L ^2.

式中、Ｒ^１１、Ｒ^１２、Ｒ^１４及びＲ^１５はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよく、Ｒ^１１、Ｒ^１２、Ｒ^１４及びＲ^１５の少なくとも２つは水素原子以外の原子又は基である。
Ｘ^１及びＸ^２はそれぞれ独立に、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、ヒドロキシ基、アルコキシ基、アリールオキシ基、アルキルチオ基、アリールチオ基、エテニル基、又はエチニル基を表し、これらは置換基を有していてもよく、Ｘ^１及びＸ^２は互いに連結して環を形成してもよい。
Ｒ^３１〜Ｒ^３５はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、シアノ基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよく、Ｒ^３１〜Ｒ^３５のいずれか一つは水素原子である。
Ｒ^５１及びＲ^５２はそれぞれ独立に、アルキル基、アリール基、ヘテロアリール基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。
Ｑ^１及びＱ^２はそれぞれ独立に、芳香族炭化水素環又は芳香族ヘテロ環を表し、これらは置換基を有していてもよい。
Ｌ^１及びＬ^２はそれぞれ独立に、式（Ｌ−１）〜式（Ｌ−４）の何れかを表す。

式中、Ｒ^１１１〜Ｒ^１１６はそれぞれ独立に、水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アミノ基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、又はアリールチオ基を表し、これらは置換基を有していてもよい。Ａは、−Ｏ−、−Ｓ−、又は−ＮＨ−を表す。<About the compound represented by the formula (6)>
The compound represented by the formula (5) is more preferably the compound represented by the following formula (6).

In the formula, R ¹¹ , R ¹² , R ¹⁴ and R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, respectively. Representing an aryloxy group, an alkylthio group, or an arylthio group, which may have a substituent ^{, at least two of R 11} , R ¹² , R ¹⁴ and R ¹⁵ are atoms or groups other than hydrogen atoms. ..
X ¹ and X ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group. It may have a substituent and X ¹ and X ² may be linked to each other to form a ring.
R ^{31 to} R ³⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, cyano group, alkoxy group, aryloxy group and alkylthio group. , Or an arylthio group, which may have a substituent, and ^{any one of R 31 to} R ³⁵ is a hydrogen atom.
R ⁵¹ and R ⁵² each independently represent an alkyl group, an aryl group, a heteroaryl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group, and these have a substituent. You may.
Q ¹ and Q ² each independently represent an aromatic hydrocarbon ring or aromatic hetero ring, which may have a substituent.
L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-.

R ¹¹ and R ¹⁵ are preferably alkyl groups, aryl groups, heterocyclic groups, ethenyl groups, ethynyl groups, and amino groups, respectively, ^{and are synonymous with R 41} and R ⁴² , that is, aryl groups. , Heterocyclic group, ethenyl group, or ethynyl group is more preferable, and aryl group, ethenyl group, ethynyl group is further preferable. From the viewpoint of improving the quantum yield, an aryl group is more preferable, and from the viewpoint of lengthening the wavelength, an ethenyl group and an ethynyl group are more preferable. In the case of an aryl group, it is preferable to have at least one substituent at the ortho-position or meta-position of the aryl group, and it is more preferable to have at least one substituent at the ortho-position. The number of substituents substituted with the aryl group is preferably 1 to 3, more preferably 2 or 3. The substituent to be substituted with the aryl group is preferably an alkyl group, more preferably a methyl group, an isopropyl group, or a t-butyl group, and even more preferably a methyl group.

<Specific examples of compounds represented by formulas (1) to (6)>
Specific examples of the compounds represented by the formulas (1) to (6) are described below. Me indicates a methyl group, Et indicates an ethyl group, and iPr indicates an isopropyl group.

The labeled particles may be labeled particles containing at least one energy donor compound, at least one energy acceptor compound, and particles, in which case at least one of the energy donor compound and the energy acceptor compound. However, any compound represented by the above formula (1) may be used.

In another example of the present invention, the luminescent particle contains a compound represented by the formula (1) as either an energy donor compound or an energy acceptor compound, and is either an energy donor compound or an energy acceptor compound. On the other hand, it contains a compound represented by the formula (10) described later. That is, the luminescent particles may be luminescent particles containing the compound represented by the formula (1) as the energy donor compound and the compound represented by the formula (10) as the energy acceptor compound, or the energy acceptor. Luminescent particles containing the compound represented by the formula (1) as the compound and the compound represented by the formula (10) as the energy donor compound may be used.

<Compound represented by formula (10)>

In the formula (10), m1 and m2 each independently represent an integer of 0 to 4, and any one of m1 and m2 is at least 1 or more. M represents a metalloid atom or a metal atom. R ¹ , R ² and R ³ independently represent a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group or an arylthio group. These may have substituents. Y ¹ and Y ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group, which are substituted. It may have a group, and Y ¹ and Y ² may be connected to each other to form a ring. Ar ¹¹ and Ar ¹² each independently represent an aromatic ring which may have a substituent. Z ¹ and Z ² each independently represent an aryl group, a heterocyclic group or an amino group, which may have a substituent. If m1 is 2 or more, a plurality of Z ¹ may be different each be the same group group, when m2 is 2 or more, a plurality of Z ² may each be the same group different groups.

In the formula (10), m1 and m2 each independently represent an integer of 0 to 4, and preferably m1 and m2 are both 1 or more. m1 and m2 may be the same or different integers, but are preferably the same integer. Preferably, m1 and m2 are independently 1 or 2, respectively, more preferably m1 and m2 are both 1 or both, and particularly preferably m1 and m2 are both 1.

In formula (10), M represents a metalloid atom or a metal atom, preferably a metalloid atom, and particularly preferably a boron atom.

In formula (10), R ¹ , R ² and R ³ are independently hydrogen atoms, alkyl groups, aryl groups, heterocyclic groups, ethenyl groups, ethynyl groups, acyl groups, alkoxy groups, aryloxy groups, alkylthio groups, respectively. Alternatively, it represents an arylthio group, which may have a substituent.
Preferably, R ¹ and R ² are independently aryl groups or heterocyclic groups, which may have substituents.
R ¹ and R ² may be the same or different, but are preferably the same.
R ¹ and R ² do not connect to form a ring.
Preferably, R ³ is a hydrogen atom, an alkyl group, an aryl group or a heterocyclic group, which may have a substituent. More preferably, R ³ is a hydrogen atom.

In formula (10), Y ¹ and Y ² are independently halogen atoms, alkyl groups, aryl groups, heterocyclic groups, hydroxy groups, alkoxy groups, aryloxy groups, alkylthio groups, arylthio groups, ethenyl groups, or ethynyl groups, respectively. These may have substituents, and Y ¹ and Y ² may be linked to each other to form a ring.
Preferably, Y ¹ and Y ² independently represent a halogen atom, an alkyl group, an aryl group, a hydroxy group, an alkoxy group, or an aryloxy group, which may have a substituent, Y 1 and ^{Y 2.} Y ² may be connected to each other to form a ring.
More preferably, Y ¹ and Y ² are independent halogen atoms.
More preferably, Y ¹ and Y ² are fluorine atoms.
Y ¹ and Y ² may be the same or different, but are preferably the same.

In formula (10), Ar ¹¹ and Ar ¹² each independently represent an aromatic ring which may have a substituent.
Preferably, Ar ¹¹ and Ar ¹² represent a benzene ring.

In formula (10), Z ¹ and Z ² independently represent an aryl group, a heterocyclic group or an amino group, which may have a substituent. If m1 is 2 or more, a plurality of Z ¹ may be different each be the same group group, when m2 is 2 or more, a plurality of Z ² may each be the same group different groups.
Preferably, Z ¹ and Z ² each independently represent an aryl group that may have a substituent.
More preferably, Z ¹ and Z ² independently represent a phenyl group, a naphthyl group, or an anthryl group, which may have a substituent.
Preferably, when m1 is 2 or more, a plurality of Z ¹ are the same group.
Preferably, when m2 is 2 or more, a plurality of Z ² are the same group.
The compound represented by the formula (10) preferably does not have an acidic group such as a carboxylic acid group, a phosphoric acid group or a sulfonic acid group in the molecule.

<About the compound represented by the formula (10A)>
Preferred examples of the compound represented by the formula (10) include a compound represented by the following formula (10A).

In formula (10A), Y ¹ and Y ² are independently halogen atoms, alkyl groups, aryl groups, heterocyclic groups, hydroxy groups, alkoxy groups, aryloxy groups, alkylthio groups, arylthio groups, ethenyl groups, or ethynyl groups, respectively. , And these may have substituents. Examples of the substituent include the substituents described in the substituent group A.
Preferably, Y ¹ and Y ² each independently represent a halogen atom.
Particularly preferably, Y ¹ and Y ² are fluorine atoms.

In formula (10A), R ³ represents a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, or an acyl group, which may have a substituent.
Preferably, R ³ is a hydrogen atom, an alkyl group, an aryl group or a heterocyclic group, which may have a substituent.
More preferably, R ³ is a hydrogen atom.

In formula (10A), Ar ³ and Ar ⁴ each independently represent an aryl group or a heterocyclic group, which may have a substituent. Examples of the substituent include the substituents described in the substituent group A.

In formula (10A), R ^{34 to} R ⁴¹ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, acyl group, alkoxy group, aryloxy group, alkylthio group, respectively. It represents an arylthio group or an amino group, which may have a substituent. Examples of the substituent include the substituents described in the substituent group A.

In formula (10A), preferably ^{at least one or more of R 34 to} R ⁴¹ is an aryl group which may have a substituent.
More preferably, ^{at least one or more of R 34 to} R ³⁷ are aryl groups which may have a substituent, and at least one or more of ^{R 38 to} R ^{41 may have a substituent.} A good aryl group.

式（１１）中、Ｒ^２０１〜Ｒ^２０５は水素原子、ハロゲン原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、アシル基、アルコキシ基、アリールオキシ基、アルキルチオ基、アリールチオ基、又はアミノ基であり、Ｒ^２０１及びＲ^２０５の少なくとも一方は水素原子以外の原子又は基である。Ｒ^２０１とＲ^２０２は互いに連結して環を形成してもよく、Ｒ^２０２とＲ^２０３は互いに連結して環を形成してもよく、Ｒ^２０３とＲ^２０４は互いに連結して環を形成してもよく、Ｒ^２０４とＲ^２０５は互いに連結して環を形成してもよい。More preferably, ^{at least one or more of R 34 to} R ⁴¹ is a group represented by the formula (11). More preferably, ^{at least one or more of R 34 to} R ³⁷ is a group represented by the formula (11), and ^{at least one or more of R 38 to} R ⁴¹ is a group represented by the formula (11). be.

In formula (11), R ^{201 to} R ²⁰⁵ are hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, acyl group, alkoxy group, aryloxy group, alkylthio group, arylthio group, Alternatively, it is an amino group, and ^{at least one of R 201} and R ²⁰⁵ is an atom or group other than a hydrogen atom. R ²⁰¹ and R ²⁰² may be connected to each other to form a ring, R ²⁰² and R ²⁰³ may be connected to each other to form a ring, and R ²⁰³ and R ²⁰⁴ may be connected to each other to form a ring. R ²⁰⁴ and R ²⁰⁵ may be connected to each other to form a ring.

式（１２）中、Ｒ^１０１は、水素原子、アルキル基、アリール基、ヘテロ環基、エテニル基、エチニル基、又はアシル基を表し、これらは置換基を有していてもよい。上記置換基としては、置換基群Ａに記載の置換基が挙げられる。Ａｒ^１０１はアリール基、又はヘテロ環基を表し、これらは置換基を有していてもよい。上記置換基としては、置換基群Ａに記載の置換基が挙げられる。Ａｒ^１０１とＲ^１０１は互いに連結して環を形成してもよい。
式（１０Ａ）で示される化合物は、分子内に、カルボン酸基、リン酸基、スルホン酸基などの酸性基を有さないことが好ましい。According to another preferred embodiment, ^{at least one or more of R 34 to} R ⁴¹ is a group represented by the formula (12). More preferably, ^{at least one or more of R 34 to} R ³⁷ is a group represented by the formula (12), and ^{at least one or more of R 38 to} R ⁴¹ is a group represented by the formula (12). be.

In formula (12), R ¹⁰¹ represents a hydrogen atom, an alkyl group, an aryl group, a heterocyclic group, an ethenyl group, an ethynyl group, or an acyl group, which may have a substituent. Examples of the substituent include the substituents described in the substituent group A. Ar ¹⁰¹ represents an aryl group or a heterocyclic group, which may have a substituent. Examples of the substituent include the substituents described in the substituent group A. Ar ¹⁰¹ and R ¹⁰¹ may be connected to each other to form a ring.
The compound represented by the formula (10A) preferably does not have an acidic group such as a carboxylic acid group, a phosphoric acid group, or a sulfonic acid group in the molecule.

<Specific example of the compound represented by the formula (10) or the formula (10A)>
Specific examples of the compound represented by the formula (10) or the formula (10A) are described below. Me represents a methyl group, Bu represents an n-butyl group, and Ph represents a phenyl group.

<Specific examples of combinations of energy donor compounds and energy acceptor compounds>
Specific examples of the combination of the energy donor compound and the energy acceptor compound are described below.

Regarding the selection of energy donor compounds and energy acceptor compounds, compounds with short absorption wavelengths are energy donor compounds, compounds with long absorption wavelengths are energy acceptor compounds, and light emission and energy acceptor compounds of energy donor compounds. If the absorptions of the compounds overlap even a little, it may be possible to use them in the particles of the present invention. It is preferable that the maximum absorption wavelength of the energy acceptor compound is on the long wavelength side of about 10 to 100 nm from the absorption wavelength of the energy donor compound. It is more preferable that the maximum absorption wavelength of the energy acceptor compound is 10 to 70 nm longer than the absorption wavelength of the energy donor compound.

How long the emission of the energy donor compound is emitted at the long wavelength of absorption (the magnitude of the Stokes shift) differs depending on the compound, so it cannot be said unconditionally. Since there is a maximum emission and an emission spectrum exists from there to about +100 nm, it is assumed that an energy transfer system can be realized by using an acceptor compound having absorption in the vicinity thereof.

The absorption wavelength of each compound can be predicted not only by synthesizing the compound but also by calculation by Gaussian or the like, and from the relationship of the calculated values, the combination of the energy donor compound and the energy acceptor compound. Can also be estimated.

In the present invention, the magnitude of the Stokes shift is preferably 25 nm or more, more preferably 30 nm or more, even more preferably 35 nm or more, still more preferably 40 nm or more, still more preferably 45 nm or more. It is particularly preferably 50 nm or more, and most preferably 60 nm or more. The upper limit of the size of the Stokes shift is not particularly limited, but is generally 150 nm or less.

<Amount of compounds represented by formulas (1) to (6) used>
The content of the compound represented by the formulas (1) to (6) with respect to the particles used in the present invention (that is, the particles before adding the compound represented by the formulas (1) to (6)) is the content of the present invention. It is not particularly limited as long as the effect is not impaired, but is preferably 0.5 μmol / g to 400 μmol / g, more preferably 1 μmol / g to 300 μmol / g, and further preferably 2 μmol / g to 200 μmol / g. Particularly preferably, it is 3 μmol / g to 100 μmol / g.

The content of the compound represented by the formulas (1) to (6) with respect to the particles used in the present invention (that is, the particles before adding the compounds represented by the formulas (1) to (6)) is the content of the present invention. It is not particularly limited as long as the effect is not impaired, but is preferably 0.1% by mass to 30% by mass, more preferably 0.2% by mass to 20% by mass, and further preferably 0.3% by mass to 10% by mass. %, Especially preferably 0.4% by mass to 8% by mass.

In the luminescent particles of the present invention, at least one compound represented by the formulas (1) to (6) is used, but even if two or more kinds of compounds represented by the formulas (1) to (6) are used. good. When two or more compounds represented by the formulas (1) to (6) are used, the total amount is preferably within the above range.

When a combination of the energy donor compound and the energy acceptor compound is used, the molar ratio of the energy donor compound and the energy acceptor compound is preferably 1:10 to 20: 1, preferably 1:10 to 10: 1. Is more preferable, and 1: 5 to 10: 1 is more preferable.

When at least one compound represented by the formula (1) is used as the energy donor compound and at least one compound represented by the formula (1) is used as the energy acceptor compound, two or more kinds are used as the energy donor compound. The compound represented by the formula (1) may be used, or two or more kinds of compounds represented by the formula (1) may be used as the energy acceptor compound. In the above case, it is preferable that the total amount of the compounds represented by the formula (1) used is within the above range.

<Method for producing compounds represented by formulas (1) to (6)>
The compounds represented by the formulas (1) to (6) can be produced, for example, by the synthetic scheme shown in Examples described later.

As an example, an outline of the synthesis of compound (1) is shown below. To a mixture of 3,5-bis (trifluoromethyl) benzaldehyde and dichloromethane, 3-ethyl-2,4-dimethylpyrrole and trifluoroacetic acid are added while cooling with water, the mixture is stirred at room temperature, and chloranyl is added while cooling with water. After stirring at room temperature, diisopropylethylamine was added dropwise while cooling with water, and the mixture was stirred at room temperature. Then, boron trifluoride diethyl ether complex was added dropwise while cooling with water, and the mixture was stirred at room temperature to carry out the reaction. (1-A) can be synthesized. Subsequently, compound (1-A), 2,4,6-trimethylbenzaldehyde, and dehydrated toluene are mixed and stirred at room temperature. A compound (a compound (1) of piperidine and p-toluenesulfonic acid monohydrate was added and stirred while distilling off the solvent, and after allowing to cool, dehydrated toluene was added and the solvent was stirred while stirring to carry out the reaction. 1) can be manufactured.

As another example, compound (5) is compound (5-A) starting from 2,3,5,6-tetrafluorobenzaldehyde and 2,4-dimethylpyrrole according to the synthetic scheme in the examples described below. , Compound (5-B), and compound (5-C).

Compound (1) and compound (5) are within the definition of the compound represented by the formula (1). For compounds represented by the formula (1) other than the compound (1) and the compound (5), the compound used in the reaction is replaced with a compound having a substituent corresponding to the desired compound represented by the desired formula (1). By doing so, it can be manufactured.

<Method for producing the compound represented by the formula (10)>
The compound represented by the formula (10) can be produced, for example, by the synthetic scheme shown below.

^{The definitions of R 1} and Z ¹ in the above synthesis scheme are synonymous with the definitions ^{of R 1} and Z ¹ in the formula (10).
Compound A-30 can be synthesized by reacting compound A-10 with compound A-20 according to the method described in Macromolecules 2010, 43, 193-200. Then, Compound A-30, ^wherein: Z 1 -B _(OH) compounds represented by _2, and added cesium fluoride (CsF) in a mixed solution of water and dimethoxyethane (DME), evacuated, and purged with nitrogen Repeat degassing. Palladium acetate (Pd (OAc) ₂ ) and 2-dicyclohexylphosphino-2', 6'-dimethoxybiphenyl (SPhos) are added, the temperature is raised, and the reaction is carried out under reflux for a predetermined time (for example, 2 to 24 hours). By allowing the compound D-10 to be produced, the compound D-10 can be produced.

Compound D-10 is within the definition of the compound represented by the formula (10). For even compounds compounds represented by the D-10 other than the formula (10), Compound A-10 Compound A-20, and the ^formula: Z 1 -B _(OH) any one or more compounds of the compounds represented by the ₂ Can be produced by replacing with the corresponding compound.

(First particle with label)
The labeled first particle in the present invention exhibits a high quantum yield and high brightness by containing the compound represented by the formula (1).
The maximum excitation wavelength of the first labeled particle is the wavelength with the highest fluorescence intensity in the excitation spectrum. The fluorescence maximum wavelength of the first particle having a label is the wavelength having the highest fluorescence intensity in the fluorescence spectrum. Further, the excitation spectrum indicates the excitation wavelength dependence of the fluorescence labeling intensity, and the fluorescence spectrum indicates the fluorescence wavelength dependence of the fluorescence intensity.
The maximum excitation wavelength of the labeled first particle is preferably 640 nm to 900 nm, more preferably 640 nm to 800 nm, and even more preferably 650 nm to 750 nm.
The maximum fluorescence wavelength of the labeled first particle is preferably 660 nm to 900 nm, more preferably 660 nm to 800 nm, and even more preferably 670 nm to 750 nm.
The fluorescence intensity of the first particle having a label is the fluorescence intensity when measured under certain measurement conditions, and is generally used for relative comparison because it depends on the measurement conditions.

The excitation maximum wavelength, fluorescence maximum wavelength, and fluorescence intensity of the first particle having a label can be measured using a commercially available fluorescence spectrophotometer, for example, a fluorescence spectrophotometer RF-5300PC manufactured by Shimadzu Corporation. Can be measured using.

The quantum yield of the labeled first particle is the ratio of the number of photons emitted as fluorescence to the number of photons absorbed by the labeled first particle.
The quantum yield of the labeled first particle is preferably 0.25 or more, more preferably 0.30 or more, still more preferably 0.40 or more. The upper limit of the quantum yield is not particularly limited, but is generally 1.0 or less.
The quantum yield of the labeled first particle can be measured using a commercially available quantum yield measuring device, for example, using an absolute PL quantum yield measuring device C9920-02 manufactured by Hamamatsu Photonics. Can be measured.

<Method for producing the first particle with a label>
The method for producing the first particles having a label is not particularly limited, but the first particles can be produced by mixing at least one compound represented by the formula (1) with the particles. For example, by adding the compound represented by the formula (1) to particles such as latex particles, the first particles having a label can be prepared. More specifically, by adding a solution containing the compound represented by the formula (1) to a solution of particles containing any one or more of water and a water-soluble organic solvent (tetrahydrofuran, methanol, etc.) and stirring the mixture. A first particle with a label can be produced.

In the present invention, a dispersion liquid containing the first particles having the above-mentioned label may be prepared.
The dispersion can be produced by dispersing the labeled first particles in a dispersion medium. Examples of the dispersion medium include water, an organic solvent, or a mixture of water and an organic solvent. As the organic solvent, alcohols such as methanol, ethanol and isopropanol, ether solvents such as tetrahydrofuran and the like can be used.
The solid content concentration of the labeled first particles in the dispersion is not particularly limited, but is generally 0.1 to 20% by mass, preferably 0.5 to 10% by mass, and more preferably 1. ~ 5% by mass.

(Modification of the first particle with the label by the first binding agent)
The method for immobilizing the first binding substance on the labeled first particles is described in, for example, Japanese Patent Application Laid-Open No. 2000-206115 and the protocol attached to Thermo Fishers (registered trademark) polystyrene microspheres F8813. Any known method for preparing a reagent for immunoagglutination reaction can be used. Further, as a principle of immobilizing an antibody on particles as a binding substance, either the principle of physical adsorption or the principle of chemical bond by covalent bond can be adopted. Blocking agents (ie, first blocking agents) that cover the surface of particles that are not coated with the antibody after the antibody is immobilized on the particles include, for example, albumin (BSA, etc.), skim milk, casein, soybean-derived components, fish-derived. Commercially available blocking agents for immune reactions containing the above-mentioned substances or substances having the same properties as the above-mentioned substances can be used. These blocking agents can also be pretreated with heat, acid, alkali or the like, if necessary, such as partial denaturation. Further, as the first blocking agent, an antibody (globulin) having no binding property to progesterone, a protein not used in the test area (Protein A, Protein G), or the like can also be used.

Specific methods for immobilizing the antibody on the particles are illustrated below. An antibody solution adjusted to a concentration of 0.01 to 20 mg / mL is added to a solution dispersed so that the solid content concentration of the particles is 0.1 to 10% by mass, and the mixture is mixed. Stirring is continued for 5 minutes to 48 hours under the condition of a temperature of 4 to 50 ° C. Then, the particles and the solution are separated by centrifugation or other methods to sufficiently remove the antibody contained in the solution that has not bound to the particles. Then, the operation of washing the particles with the buffer solution is repeated 0 to 10 times. After the operation of mixing the particles and the antibody to bind the antibody to the particles, components not involved in the antigen-antibody reaction, preferably proteins, more preferably globulin, albumin, Block Ace®, skim milk and casein. It is desirable to protect the part of the particle surface where the antibody is not bound by using a blocking agent such as.

When immobilizing an antigen, an antibody or the like on particles, a stabilizer can be added as needed. The stabilizer is not particularly limited as long as it stabilizes antigens and antibodies such as synthetic polymers such as sucrose and polysaccharides or natural polymers, such as Immunoassay Stabilizer (Advanced Biotechnologies Inc (ABI)). Commercially available stabilizers can also be used.

The labeled first particles, modified with the first binding agent, are included in the kit of the invention, preferably in a container, eg, a cup, which is part of the kit. In this case, the progesterone in the biological sample and the first binding substance can be bound by injecting the biological sample containing progesterone into a container containing the first labeled particles, mixing and stirring.

(Second binding substance)
The unlabeled second particle in the present invention is modified with a second binding agent that does not have specific binding to progesterone. The second binding substance is, for example, a compound having no specific binding property to progesterone, such as a binding substance (antibody) or a protein (Protein A, Protein G) that binds to the binding substance (antibody). Moreover, the compound is not particularly limited as long as it does not have an affinity for the first binding substance, and any compound can be preferably used. For example, when the second binding agent is an antibody, by cell fusion using an antiserum prepared from the serum of an immunized animal, an immunoglobulin fraction purified from the antiserum, and spleen cells of an immunized animal. The obtained monoclonal antibody or a fragment thereof [for example, F (ab') 2, Fab, Fab', or Fv] can be used. Preparation of these antibodies can be carried out by a conventional method. Further, the antibody may be modified as in the case of a chimeric antibody or the like, and a commercially available antibody or an antibody prepared by a known method from animal serum or culture supernatant can be used.

A method for immobilizing a second binding substance such as an antibody on particles is described in, for example, Japanese Patent Application Laid-Open No. 2000-206115 and a protocol attached to Molecular Probes Co., Ltd. FluoroSpheres (registered trademark) polystyrene microspheres F8813. Any known method for preparing a reagent for an immunoaggregation reaction can be used. Further, as a principle of immobilizing an antibody on particles as a binding substance, either the principle of physical adsorption or the principle of chemical bond by covalent bond can be adopted. Known substances, such as BSA, skim milk, casein, soybean-derived components, fish-derived components, polyethylene glycol, and the like, are known substances as blocking agents that cover the surface of particles not coated with the antibody after the antibody is immobilized on the particles. And commercially available blocking agents for immune reactions containing substances having the same properties as these can be used. These blocking agents can also be pretreated with heat, acid, alkali or the like, if necessary, such as partial denaturation.

(First particle and second particle)
Regarding the usage ratio of the first particle and the second particle, the mass ratio of the second particle to the first particle is generally 1 to 20, preferably 1 to 10, and 2 to 8 Is more preferable, and 4 to 6 is even more preferable.

In the present invention, the average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. The average particle size is large.
The average particle size of the first particles is preferably 70 to 130 nm, more preferably 80 to 120 nm, and even more preferably 85 to 110 nm.
The average particle size of the second particle is preferably 130 to 300 nm, more preferably 135 to 260 nm, and even more preferably 140 to 200 nm.

The methods for measuring the average particle size include optical microscopy, confocal laser microscopy, electron microscopy, interatomic force microscopy, static light scattering, laser diffraction, dynamic light scattering, centrifugal sedimentation, and electricity. Pulse measurement methods, chromatography methods, ultrasonic attenuation methods, etc. are known, and devices corresponding to each principle are commercially available. Among these measuring methods, it is preferable to measure the average particle size of fluorescent particles by using a dynamic light scattering method because of the particle size range and ease of measurement. Commercially available measuring devices using dynamic light scattering include Nanotrack UPA (Nikkiso Co., Ltd.), dynamic light scattering particle size distribution measuring device LB-550 (HORIBA, Ltd.), and concentrated particle size analyzer. FPAR-1000 (Otsuka Electronics Co., Ltd.) and the like can be mentioned. In the present invention, the average particle size is determined as the median diameter (d = 50) measured at 25 ° C. under the conditions of viscosity 0.8872 CP (however, 1 Pa · s = 1000 cP) and refractive index of water 1.330. And.

The first particle (labeled particle) and the second particle used in the present invention may be stored in a dry state and used by mixing with a biological sample containing progesterone at the time of measurement. When the first particle and the second particle are stored in a solution state, the particles may agglomerate or fuse with each other to increase the size, and the measurement accuracy may change. Particles and second particles can be stored in a dry state. Particles stored in a dry state are also called dry particles. The dry particles are preferably 30% by mass or less, more preferably 25% by mass or less, still more preferably 20% by mass, as the mass of water (water content) with respect to the mass of the solid content of the particles containing the labeling substance containing no water. Particles in which the amount of water contained is removed until the mass of water is as follows. The means for drying is not particularly limited, and for example, known drying means such as a drying method using a dehumidifying agent, a vacuum drying method, and a freeze-drying method can be used. In the present invention, the first particles and the second particles may be dried separately to obtain dried particles, or the first particles and the second particles are mixed in a solution state at a desired mass ratio. It may be dried later to obtain dried particles.

(Flow path)
The kit of the present invention includes a flow path for flowing the first particle and the second particle. In the present invention, a mixed solution of a biological sample that may contain progesterone, a first particle having a label, and a second particle without a label is applied onto a substrate and applied to a flow path. Can be deployed. The flow path is not particularly limited as long as it is a flow path through which the biological sample, the first particle having a label, and the second particle flow down to the reaction site. Preferred flow path embodiments include a spotting port for instilling a biological sample solution containing a labeled first particle and a second particle, a metal thin film as a reaction site on which a third binding substance is immobilized, and the like. In addition, there is a flow path beyond the metal thin film, and the biological sample has a structure that allows it to pass over the metal thin film. Preferably, the suction port can be provided on the side of the metal thin film opposite to the spotting port.

(substrate)
In the present invention, in order to achieve highly sensitive measurement, it is preferable to adopt a measurement method for detecting surface plasmon fluorescence (SPF), which will be described later. As the substrate in this case, it is preferable to use a substrate having a metal film on the surface. The metal constituting the metal film is not particularly limited as long as it can cause surface plasmon resonance. Free electron metals such as gold, silver, copper, aluminum, and platinum are preferred, with gold being particularly preferred. When gold is used, the detection region described later is on the gold film. The above metals can be used alone or in combination. Further, in consideration of the adhesiveness to the substrate, an intervening layer made of chromium or the like may be provided between the substrate and the layer made of metal. The film thickness of the metal film is arbitrary, but is preferably 1 nm or more and 500 nm or less, and particularly preferably 10 nm or more and 200 nm or less. If it exceeds 500 nm, the surface plasmon phenomenon of the medium cannot be sufficiently detected. When an intervening layer made of chromium or the like is provided, the thickness of the intervening layer is preferably 0.1 nm or more and 10 nm or less.

The metal film may be formed by a conventional method, for example, by a sputtering method, a vapor deposition method, an ion plating method, an electroplating method, an electrolytic plating method, or the like, but a mixture of the substrate material and the metal film is performed. In order to provide the layer and improve the adhesion of the metal film, it is preferable to prepare the metal film by a plating method. In this case, the thickness of the mixed layer of the substrate material and the metal film is not particularly limited as long as sufficient adhesion can be ensured, but is preferably 10 nm or less.

The metal film is preferably arranged on the substrate. Here, "arranged on the substrate" means that the metal film is arranged so as to be in direct contact with the substrate, and that the metal film is not in direct contact with the substrate and is placed through another layer. Including the case where it is arranged. Examples of the substrate material that can be used in the present invention include optical glass such as BK7 (borosilicate glass), which is a kind of general optical glass, or synthetic resin, specifically, polymethylmethacrylate and polyethylene terephthalate. A material made of a material transparent to laser light such as polycarbonate or a cycloolefin polymer can be used. Such a substrate is preferably made of a material that does not exhibit anisotropy with respect to polarized light and has excellent workability.

A preferred embodiment of the substrate for SPF detection includes a substrate in which a gold film is formed on polymethylmethacrylate (PMMA) by a sputtering method or the like.

The substrate comprises a detection region having a substance that has a binding property to the first binding substance.

A method for immobilizing a substance having a binding property to the first binding substance on a substrate is described in, for example, Tech Notes Vol. 2-12 provided by Nunc, and is described in general ELISA (Enzyme-). Linked ImmunoSorbent Assay) Any known method for preparing a reagent can be used. Further, the surface may be modified by arranging a self-assembled monolayer (SAM) or the like on the substrate. As a principle of immobilizing a substance having a binding property to the first binding substance on a substrate, either the principle of physical adsorption or the principle of chemical bonding by covalent bond can be adopted. A substance known as a blocking agent that covers a substrate surface that is not coated with a substance having a binding property to the first binding substance after fixing a substance having a binding property to the first binding substance to the substrate. For example, BSA, skim milk, casein, soybean-derived components, fish-derived components, polyethylene glycol and the like, and commercially available blocking agents for immune reactions containing these substances and substances having the same properties as these can be used. These blocking agents can also be pretreated with heat, acid, alkali or the like, if necessary, such as partial denaturation.

(Detection area <test area>)
In the present invention, a test area for detecting the presence or absence of progesterone in a biological sample can be provided on the substrate. In this test area, it is possible to quantify the antigen by a method in which only the antigen-bound label cannot be bound, only the non-antigen-bound label is captured, and the amount of the antigen-bound label is calculated. It becomes. This detection method is called the competition method, but here, the substrate related to the competition method will be described.

The test area of the substrate preferably has sites that react with binding substances (eg, antibodies) present on the labeled particles. As a preferred embodiment of the present invention, it is preferable to have progesterone present in the biological sample on the test area of the substrate. In this case, a test area can be prepared by reacting progesterone and BSA in the presence of a condensing agent to prepare a progesterone / BSA conjugate, and adsorbing this conjugate on the test area. The progesterone-BSA conjugate, which is the substance to be measured, is dissolved in a buffer solution, instilled on the substrate, left for a certain period of time, and then the supernatant is aspirated and dried. It is possible to combine with.

(Reference area <control area>)
In the present invention, in order to suppress the influence of the measurement environment, particularly the measurement temperature as much as possible, the control area is provided on the substrate, and the information of the test area is standardized by the information of the control area. It is possible to keep it low. The control area is preferably designed so that it can bind to all labels, independent of the amount of progesterone present in the biological sample used. It is preferable to have an antibody that interacts with all the antibodies present on the labeled particles. By designing in this way, the information in the test area is standardized by the information in the control area. It is possible to cancel the effect and obtain a result that is always accurate and unaffected by the measurement environment.

The preferable antibody to be present in the control area has a function of recognizing a binding substance (for example, an antibody) existing on the labeled particles, and if the antibody is derived from a mouse, it is preferably an anti-mouse antibody, and the labeled particles. If the above antibody is derived from goat, it is preferably an anti-goat antibody. The antibodies on these control areas can be dissolved in a buffer solution, instilled on the substrate, left for a certain period of time, and then the supernatant can be sucked and dried to bind to the substrate. ..

(Blocking agent)
For example, in the competitive method, there are biological samples that are negative in response to not only negative biological samples that do not contain progesterone but also positive biological samples that contain progesterone. The solution is recognized as an issue. The cause of such false negatives has not been clarified, but we do not want them to bind to each other due to the non-specific interaction between the surface of the labeled particles not covered by the antibody and the detection area (test area). It is believed that one of the causes is the presence of labeled particles. Further, even when the same substance as the substance existing on the test area is present on the surface of the labeled particle, if a released antibody or the like is present in the biological sample, the antibody is a substance existing on the test area. By binding to either the substance on the surface of the labeled particle, it may be detected as negative even when a positive biological sample containing progesterone is measured.
Generally, blocking with BSA is used to suppress non-specific adsorption on a solid phase surface (for example, a labeled particle surface or a gold film surface of a substrate).

(antibody)
In the present invention, the antibody can be used regardless of its animal species, subclass, and the like. For example, the antibodies that can be used in the present invention are antibodies derived from organisms such as mice, rats, hamsters, goats, rabbits, sheep, cows, and chickens that can cause an immune reaction, specifically, mouse IgG and mouse IgM. , Rat IgG, Rat IgM, Hamster IgG, Hamster IgM, Rabbit IgG, Rabbit IgM, Goat IgG, Goat IgM, Sheep IgG, Sheep IgM, Bovine IgG, Bovine IgM, Trial IgY, etc. It is possible. A fragmented antibody is a molecule derived from a complete antibody having at least one antigen binding site, specifically F (ab') ₂ , Fab, Fab', Fv, or the like. These fragmented antibodies are molecules obtained by enzymatic or chemical treatment, or by genetic engineering techniques.

(Other elements of the kit)
The kit of the present invention is used for a method for measuring progesterone. The kit of the present invention includes a first particle having a label, a second particle without a label, a flow path for flowing the first particle and the second particle, and a first binding substance. Includes a sensor chip that includes a substrate with a substance that is binding to. The kit of the present invention may further include various devices or devices used for measuring progesterone, such as surface plasmon excitation devices and fluorescence measurement devices. Further, as an element of the kit, a sample containing a known amount of progesterone, an instruction manual, and the like may be included.

[Measurement method of progesterone]
Further, according to the present invention
(I) (a) Labeled first particles modified with a first binding agent having a specific binding property to progesterone.
(B) An unlabeled second particle modified with a second binding agent that does not have specific binding to progesterone.
(C) A test sample containing progesterone and
To obtain a mixture by mixing
(Ii) A step of applying the mixture obtained in step (i) onto a substrate, and
(Iii) A step of capturing the first binding substance at a reaction site on a substrate having a substance having a binding property to the first binding substance.
(Iv) A step of detecting a labeled first particle modified with a first binding agent captured on the reaction site.
A method for measuring progesterone, including
The labeled first particle contains at least one compound represented by the formula (1) described herein and the particle.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. ,
A method for measuring progesterone is provided.

In the present invention, progesterone is measured by detecting the first labeled particle modified with the first binding agent captured on the reaction site.
The measurement in the present invention is interpreted as the broadest concept as long as it is a measurement of the amount of progesterone. As a specific embodiment of the measurement method, a competitive method is preferable.

The case of quantifying progesterone will be described below.
In the competitive method, first, a biological sample containing progesterone and anti-progesterone antibody-labeled fluorescent particles are brought into contact with a progesterone immunoassay substrate on which a progesterone / albumin conjugate is immobilized. In the absence of progesterone in the biological sample, the anti-progesterone antibody-labeled fluorescent particles and the progesterone on the substrate (ie, the progesterone in the progesterone-albumin conjugate) cause an antigen-antibody reaction on the substrate. On the other hand, when progesterone is present in the biological sample, an antigen-antibody reaction occurs between the progesterone in the biological sample and the anti-progesterone antibody-labeled fluorescent particles, and the anti-progesterone antibody-labeled fluorescent particles and the progesterone on the substrate (that is, that is) , Biliary acid in progesterone-albumin conjugate) is inhibited. After completion of the above reaction, the anti-progesterone antibody-labeled fluorescent particles that did not bind to albumin on the substrate are removed. Then, by detecting the degree of formation of an immune complex on the substrate (that is, a complex of anti-progesterone antibody-labeled fluorescent particles and progesterone in a progesterone-albumin-albumin conjugate on the substrate) as fluorescence intensity, in a biological sample. It is possible to measure the concentration of progesterone and the like.

As the measurement form of fluorescence in the competitive method, either plate reader measurement or flow measurement can be adopted, and for example, the measurement can be performed by the following method. A plurality of samples having a known amount of progesterone having different progesterone concentrations are prepared in advance, and this sample and anti-progesterone antibody-labeled fluorescent particles are mixed in advance. The mixture is brought into contact with the region where the progesterone / albumin conjugate is immobilized. Fluorescence signals from the region where the progesterone albumin conjugate is immobilized are measured as multiple fluorescence signals while the mixture is in contact with the conjugate at specific time intervals. From these plurality of fluorescence signals, the time change (slope) of the amount of fluorescence is obtained at each progesterone concentration. This time change is plotted on the Y-axis and the progesterone concentration is plotted on the X-axis, and a relational expression of the progesterone concentration with respect to the time change of the fluorescence amount is obtained by using an appropriate fitting method such as the least squares method. Based on the relational expression thus obtained, the amount of progesterone contained in the biological sample can be quantified by using the result of the time change of the fluorescence amount using the biological sample to be inspected.

The amount of progesterone is preferably quantified in a short time. Specifically, it is preferably performed within 10 minutes, more preferably within 8 minutes, and further preferably within 6 minutes. For this quantification time, the sample and the anti-progesterone antibody-labeled fluorescent particles were subjected to progesterone by using the relational expression between the time change of the amount of fluorescence and the progesterone concentration obtained in advance by using an appropriate fitting method such as the minimum square method. It includes the time to convert the amount of progesterone contained in the biological sample based on the result of the time change of the amount of fluorescence using the biological sample to be tested after contacting the detection region where the albumin conjugate is immobilized. Is preferable.

(Surface plasmon fluorescence measurement)
The method for detecting a label such as fluorescence in the present invention is not particularly limited, but for example, a device capable of detecting fluorescence intensity, specifically, a microplate reader or fluorescence detection (SPF) by surface plasmon excitation is used. It is preferable to detect the fluorescence intensity using a biosensor or the like. Preferably, fluorescence detection by surface plasmon resonance can obtain labeling information related to the amount of progesterone.

The form of fluorescence measurement may be plate reader measurement or flow measurement. The fluorescence detection method by surface plasmon excitation (SPF method) can measure with higher sensitivity than the fluorescence detection method by epi-emission excitation (epi-radiation fluorescence method).

Examples of the surface plasmon fluorescence (SPF) biosensor include an optical waveguide formed from a material that transmits excitation light having a predetermined wavelength, as described in Japanese Patent Application Laid-Open No. 2008-249361, and one of the optical waveguides. An optical system in which a metal film formed on the surface, a light source that generates a light beam, and the light beam are passed through an optical waveguide and incident on the interface between the optical waveguide and the metal film at an incident angle that generates surface plasmons. And a sensor provided with a fluorescence detecting means for detecting the fluorescence generated by being excited by the evanescent wave enhanced by the surface plasmon can be used.

The fluorescence detection (SPF) system by surface plasmon excitation using fluorescent particles of the present invention is preferably an assay method for detecting fluorescence from a fluorescent substance depending on the amount of progesterone immobilized on a metal film on a substrate. This is a method different from the so-called latex agglomeration method, in which, for example, a change in optical transparency is detected as turbidity due to the progress of the reaction in a solution. In the latex agglutination method, the antibody-sensitized latex in the latex reagent and the antigen in the biological sample are bound and aggregated by an antibody reaction. This agglomerate increases with time, and this is a method of quantifying the antigen concentration from the change in absorbance per unit time obtained by irradiating the agglutinate with near-infrared light. The present invention can provide a very simple method for detecting progesterone as compared with the latex agglutination method.

(Standardization)
Further, the method of the present invention is a labeling particle-related labeling information acquisition step of acquiring labeling information related to the amount of labeled particles; and a labeling acquired in the progesterone-related labeling information acquisition step of acquiring labeling information related to the amount of progesterone. A method including a standardization step of standardizing the information based on the labeling information acquired in the labeling particle-related labeling information acquisition step may also be used.

Here, a mixed solution containing a biological sample and labeled particles having a first binding substance having a binding property to progesterone is brought into contact with a substrate having a detection area (test area) and a reference area (control area). Among the steps of generating surface plasmons on the detection region and the reference region and measuring the intensity of emitted fluorescence, the step of measuring the intensity of fluorescence by the surface plasmon generated on the detection region was related to the amount of progesterone. The progesterone-related labeling information acquisition step for acquiring labeling information, and the step of measuring the intensity of fluorescence by surface plasmons generated on the reference region is the labeling particle-related labeling information acquisition step. The standardization step is to obtain the rate of increase of the fluorescence intensity acquired in these two steps in a unit time as the rate of change of the fluorescence signal value, and divide the rate of change of the signal value in the detection region by the rate of change of the signal value in the reference region. Is.

[Progesterone measurement reagent]
Further, according to the present invention, (a) the first particle having a label modified with the first binding substance having specific binding property to progesterone, and (b) having specific binding property to progesterone. A progesterone-measuring reagent comprising a second unlabeled particle modified with a second binding agent that does not, and the first particle having the above label is the formula (1) described herein. ), The first particles have an average particle size of 50 to 250 nm, the second particles have an average particle size of 70 to 500 nm, and the first particles have an average particle size of 70 to 500 nm. Provided is a progesterone measuring reagent in which the average particle size of the second particle is larger than the average particle size of one particle.
The method for measuring progesterone according to the present invention can be carried out by using the above-mentioned reagent for measuring progesterone.

Hereinafter, the present invention will be described in more detail with reference to examples of the present invention. The materials, amounts used, proportions, treatment contents, treatment procedures, etc. shown in the following examples can be appropriately changed as long as they do not deviate from the gist of the present invention. Therefore, the scope of the present invention should not be construed as limited by the specific examples shown below.

<1> Preparation of latex particles <1-1> Preparation of latex particles with an average particle size of 303 nm Ultra-pure water containing 30 g (288 mmol) of styrene (manufactured by Wako Pure Chemical Industries, Ltd.) and 1 g (12 mmol) of acrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) The mixture was suspended in 330 mL, heated to 85 ° C., an aqueous solution prepared by dissolving 1 g of potassium persulfate (KPS) (manufactured by Wako Pure Chemical Industries, Ltd.) in 25 mL was added, and the mixture was stirred at 85 ° C. and 250 rpm for 6 hours. Then, centrifugation was performed 3 times at 10,000 rpm for 6 hours to obtain latex particles. Finally, the obtained latex particles were redispersed in ultrapure water. Pure water was added to prepare a diluted solution so that the solid content concentration was 1% by mass. When determined as a median diameter (d = 50) measured at a temperature of 25 ° C. using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.), the average particle size of the latex particles was 303 nm.

<1-2> Preparation of latex particles with an average particle diameter of 220 nm 30 g (288 mmol) of styrene (manufactured by Wako Pure Chemical Industries, Ltd.) and 2 g (24 mmol) of acrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) are suspended in 330 mL of ultrapure water, and 85 The temperature was raised to ° C., an aqueous solution prepared by dissolving 1 g of potassium persulfate (KPS) (manufactured by Wako Pure Chemical Industries, Ltd.) in 25 mL was added, and the mixture was stirred at 85 ° C. and 250 rpm for 6 hours. Then, centrifugation was performed 3 times at 10,000 rpm for 6 hours to obtain latex particles. Finally, the obtained latex particles were redispersed in ultrapure water. Pure water was added to prepare a diluted solution so that the solid content concentration was 1% by mass. When determined as a median diameter (d = 50) measured at a temperature of 25 ° C. using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.), the average particle size of the latex particles was 220 nm.

<1-3> Preparation of latex particles with an average particle diameter of 151 nm 30 g (288 mmol) of styrene (manufactured by Wako Pure Chemical Industries, Ltd.) and 3 g (42 mmol) of acrylic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were suspended in 440 mL of ultrapure water, and 95 The temperature was raised to ° C., an aqueous solution prepared by dissolving 1 g of potassium persulfate (KPS) (manufactured by Wako Pure Chemical Industries, Ltd.) in 10 mL was added, and the mixture was stirred at 95 ° C. and 250 rpm for 6 hours. Then, centrifugation was performed 3 times at 10,000 rpm for 6 hours to obtain latex particles. Finally, the obtained latex particles were redispersed in ultrapure water. Pure water was added to prepare a diluted solution so that the solid content concentration was 1% by mass. When determined as a median diameter (d = 50) measured at a temperature of 25 ° C. using a particle size analyzer FPAR-1000 (Otsuka Electronics Co., Ltd.), the average particle size of the latex particles was 151 nm.

<1-4> Preparation of Latex Particles with Average Particle Diameters 129, 100, 72 nm In the preparation of latex particles with an average particle diameter of 151 nm, latex having an average particle diameter of 129, 100, 72 nm is prepared by appropriately adjusting the temperature at the time of temperature rise. Particles were prepared. The average particle size was measured in the same manner as in <1-1>.

<2> Preparation of Fluorescent Latex Particles for Comparison 100 mL of methanol was added to 100 mL of an aqueous dispersion having a solid content concentration of 2% by mass of the latex particles prepared as described above, and the mixture was stirred at room temperature for 10 minutes. On the other hand, a separately prepared fluorescent dye (comparative compound: Compound 5 described in Japanese Patent No. 3442777) was slowly added dropwise to the latex solution over 60 minutes. After the dropping was completed, the organic solvent was distilled off under reduced pressure with an evaporator, and then centrifugation and redispersion in a PBS aqueous solution were repeated three times for purification, so that the average particle size was 302 nm, 218 nm, 150 nm, 132 nm, 98 nm. Six types of comparative fluorescent latex particles having a diameter of 71 nm were prepared.

<3> Preparation of comparative fluorescent latex particles modified with anti-progesterone antibody
Fluorescent particles modified with an anti-progesterone antibody were prepared as follows.
Prepare 375 μL of a 2 mass% (solid content concentration) fluorescent latex particle aqueous solution having an average particle diameter of 150 nm prepared in <2>, and prepare a 50 mmol / L MES (2-morpholinoethanosulfonic acid, manufactured by Dojin Chemical Research Institute) buffer solution. 117 μL of (pH 6.0) was added to 5 μL of a 10 mg / mL water-soluble carbodiimide (WSC) aqueous solution, and the mixture was stirred at room temperature for 15 minutes. Then, 182.4 μL of an anti-progesterone monoclonal antibody (manufactured by GeneTex) of 0.5 mg / mL was added, and the mixture was stirred at room temperature for 1.5 hours. 37.5 μL of a 2 mol / L Glycine (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution was added, and the mixture was stirred for 15 minutes, and then the fluorescent latex particles were precipitated by centrifugation (15,000 rpm, 4 ° C., 30 minutes). The supernatant was removed, 750 μL of PBS (Phosphate Buffered Saline; Phosphate Buffered Saline; manufactured by Wako Pure Chemical Industries, Ltd.) solution (pH 7.4) was added, and the fluorescent latex particles were redistributed by an ultrasonic cleaner. Further centrifugation (15,000 rpm, 4 ° C., 15 minutes) is performed, the supernatant is removed, and then 750 μL of a PBS (pH 7.4) solution containing 1% by mass BSA is added to redistribute the fluorescent latex particles. A 1% by mass solution of anti-progesterone antibody-bound fluorescent latex particles was obtained. Progesterone antibody modification of fluorescent latex particles having an average particle size of 303 nm, 220 nm, 129 nm, 100 nm, and 72 nm was also performed in the same manner.

<4> Preparation of Latex Particles Without Fluorescent Label Latex particles modified with anti-T4 antibody were prepared as follows.
To 250 μL of a 2 mass% (solid content concentration) latex particle aqueous solution having an average particle diameter of 151 nm prepared in <1-3>, 250 μL of a 50 mmol / L MES buffer (pH 6.0) solution was added, and a 5 mg / mL anti-T4 monoclonal antibody was added. 100 μL of an antibody (Medix Anti-Thyroxine Monoclonal Antibody (6901)) was added, and the mixture was stirred at room temperature for 15 minutes. Then, 5 μL of a 10 mg / mL 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution was added, and the mixture was stirred at room temperature for 2 hours. After adding 25 μL of a 2 mol / L Glycine (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution and stirring for 30 minutes, centrifugation (15,000 rpm, 4 ° C., 15 minutes) was performed to settle the latex particles. Then, the supernatant was removed, 500 μL of PBS solution (pH 7.4) was added, and the latex particles were redispersed by an ultrasonic cleaner. Centrifuge again (15,000 rpm, 4 ° C., 15 minutes) to remove the supernatant, and then add 500 μL of PBS (pH 7.4) solution containing 1% by mass BSA to redistribute the latex particles. To prepare a 1% by mass solution of anti-T4 antibody-bound latex particles. Anti-T4 antibody modification of latex particles having other particle sizes (302 nm, 218 nm, 132 nm, 98 nm, 71 nm) was also performed in the same manner.

Similarly, using an anti-hCG antibody (Medix Anti-hCG beta monoclonal antibody (5008)), anti-hCG antibody modification was performed on latex particles having an average particle size.
As the notation of the type of mouse antibody of the unlabeled particles, the anti-T4 antibody was set to 1 and the anti-hCG antibody was set to 2, and they are shown in Table 3 below.

<5> Preparation of high-intensity fluorescent latex particles <5-1> Synthesis of compounds The terms have the following meanings.
MS: mass spectrometry
ESI: Electrospray ionization
NMR: nuclear magnetic resonance
rt: Room temperature Et: Ethyl group PL: Photoluminescence THF: tetrahydrofuran

<< Synthesis of compound (1) >>

Synthesis of Compound (1-A) 1.00 g of 3,5-bis (trifluoromethyl) benzaldehyde and 20 mL of dichloromethane were introduced into a 100 mL three-necked flask under a nitrogen atmosphere, and the mixture was stirred at room temperature. While cooling with water, 0.98 g of 3-ethyl-2,4-dimethylpyrrole was added dropwise, followed by the addition of 2 drops of trifluoroacetic acid, followed by stirring at room temperature for 30 minutes. Chloranil 1.0g was added with water-cooling, followed by stirring at room temperature for 10 minutes, cooled and added dropwise diisopropylethylamine (N ⁱ Pr ₂ Et) 3.67g while and stirred at room temperature for 15 minutes. Subsequently, 5.6 mL of boron trifluoride diethyl ether complex was added dropwise while cooling with water, and the mixture was stirred at room temperature for 30 minutes. Saturated sodium hydrogen carbonate and toluene were added dropwise, and the organic layer obtained by extraction and liquid separation was pre-dried with anhydrous sodium sulfate and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (developing solvent: hexane / ethyl acetate) and then recrystallized from methanol to obtain 1.28 g of compound (1-A).
1H NMR (CDCl 3,400MHz): δ 8.03 (s, 1H), 7.83 (s, 2H), 2.54 (s, 6H), 2.31 (q, J = 7.6Hz, 4H) , 1.21 (s, 6H), 1.00 (t, J = 7.6Hz, 6H).

Synthesis of Compound (1) 100 mg of compound (1-A), 115 mg of 2,4,6-trimethylbenzaldehyde and 5 mL of dehydrated toluene were introduced into a 100 mL three-necked flask and stirred at room temperature. Add 1 mL of piperidine and 1 piece of p-toluenesulfonic acid monohydrate (manufactured by Wako Pure Chemical Industries, Ltd., special grade reagent), stir for 1 hour while distilling off the solvent at 140 ° C., allow to cool, and then dehydrate toluene. 5 mL was added, and the mixture was stirred at 140 ° C. for 1 hour while distilling off the solvent. The reaction mixture was concentrated under reduced pressure, and the obtained crude product was purified by preparative TLC (developing solvent: hexane / ethyl acetate) and then recrystallized from methanol to obtain 71 mg of compound (1). Compounds were identified by ^{1 1} H-NMR and ESI-MS.
1H NMR (CDCl 3,400MHz): δ 8.06 (s, 1H), 7.87 (s, 2H), 7,38 (d, J = 17.2Hz, 2H), 7,32 (d, J = 17.2Hz, 2H), 6.93 (s, 4H), 2.63 (q, J = 7.6Hz, 4H), 2.44 (s, 12H), 2.30 (s, 6H), 1 .27 (s, 6H), 1.17 (t, J = 7.6Hz, 6H).
ESI-MS: [MH] ^- = 775.8

<Synthesis of compound (5)>

Synthesis of Compound (5-A) 1.16 ml of 2,4-dimethylpyrrole and 140 mL of dichloromethane were introduced into a 500 mL three-necked flask under a nitrogen atmosphere, and the mixture was stirred at room temperature. After adding 1.0 g of 2,3,5,6-tetrafluorobenzaldehyde and 1 drop of trifluoroacetic acid, the mixture was stirred at room temperature for 15 minutes. Chloranil (chloranil) 1.38 g was added and stirred at room temperature for 15 minutes, cooled and added dropwise diisopropylethylamine (N ⁱ Pr ₂ Et) 6.8mL while and stirred at room temperature for 20 minutes. Subsequently, 7.8 mL of boron trifluoride diethyl ether complex (BF ₃ · Et ₂ O) was added dropwise while cooling with water, and the mixture was stirred at room temperature for 30 minutes. 400 mL of saturated sodium hydrogen carbonate was added dropwise, and the organic layer obtained by extracting and separating with dichloromethane was pre-dried with anhydrous sodium sulfate, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (developing solvent: hexane / ethyl acetate) and then recrystallized from methanol to obtain 360 mg of compound (5-A).

Synthesis of compound (5-B)

300 mg of compound (5-A) and 8 mL of 1,1,1,3,3,3-hexafluoro-2-propanol were introduced into a 300 mL three-necked flask, and the mixture was stirred at room temperature. 409 mg of N-iodosuccinimide was introduced, and the mixture was stirred at room temperature for one and a half hours. The reaction mixture was concentrated under reduced pressure, 40 mL of methylene chloride was added, and the organic layer obtained by extraction and separation was pre-dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Ethanol was added to this crude product, and the mixture was washed and filtered to obtain 382 mg of compound (5-B).

Synthesis of compound (5-C)

278 mg of compound (5-B), 564 mg of 2,4,6-trimethylphenylboronic acid, 653 mg of cesium fluoride, and 43 mL of methoxycyclopentane were introduced into a 100 mL three-necked flask, and after degassing under reduced pressure while stirring at room temperature. , Nitrogen atmosphere. To this, 269 mg of SPhos Pd G3 (manufactured by Aldrich) was added, and the mixture was heated under reflux for 1 hour. 250 mL of ethyl acetate was added, and the organic layer obtained by extraction and liquid separation was pre-dried with anhydrous sodium sulfate, and then concentrated under reduced pressure. This composition was purified by silica gel column chromatography (developing solvent: hexane / ethyl acetate), dissolved in 5 ml of dichloromethane, and 15 ml of methanol was further added, and then dichloromethane was distilled off and reprecipitated. The precipitate was filtered to give 206 mg of compound (5-C).

Synthesis of compound (5)

50 mg of compound (5-C), 5 ml of toluene, 46 μl of 2,4,6-trimethylbenzaldehyde, 400 μl of piperidine, and one piece of p-toluenesulfonic acid were introduced into a 100 mL three-necked flask, and the mixture was heated under reflux for 1 hour under nitrogen. After adding 46 μl of 2,4,6-trimethylbenzaldehyde and heating and refluxing for 1 hour, 200 μl of piperidine was added and heated to reflux for another hour. After completion of the reaction, the mixture was concentrated under reduced pressure, the crude product was purified by silica gel column chromatography (developing solvent: hexane / toluene), dissolved in 3 ml of dichloromethane, 15 ml of methanol was added, and dichloromethane was distilled off, and reprecipitation was performed. To obtain 16 mg of compound (5). Compounds were identified by ^{1 1} H-NMR and ESI-MS.
^{1 1} H NMR (CDCl ₃ , 400 MHz): δ 7.43 (s, 1H), 7.39 (s, 1H), 7.29-7.21 (m, 1H), 6.94 (s, 4H) , 6.80 (s, 4H), 6.69 (s, 1H), 6.65 (s, 1H), 2.29 (s, 6H), 2.23 (s, 6H), 2.08 ( s, 12H), 2.03 (s, 12H), 1.33 (s, 6H).
ESI-MS: [MH] ^- = 891.4

<5-2> Preparation of high-intensity fluorescent latex particles With respect to the latex particle dispersion (latex dispersion 25 mL, solid content 500 mg) having an average particle diameter of 151 nm and a solid content concentration of 2% by mass prepared in <1-3>. THF (5 mL) was added dropwise and the mixture was stirred for 10 minutes. A THF solution (2.5 mL) containing 48 μmol / g of compound (5) was added dropwise thereto over 15 minutes. A THF solution (2.5 mL) containing 48 μmol / g of compound (1) was added dropwise thereto over 15 minutes. After completion of dropping the compound, the mixture was stirred for 30 minutes and then concentrated under reduced pressure to remove THF. Then, after centrifuging to precipitate the particles, ultrapure water was added and dispersed again to prepare a dispersion liquid of high-intensity fluorescent latex particles having a solid content concentration of 2% by mass. Further, the same operation was carried out using compound (5) instead of compound (1) to prepare a high-intensity fluorescent latex dispersion containing compound (5) and having a solid content concentration of 2% by mass.
Further, the same operation was performed using a mixture of compound (1) 24 μmol / g and compound (5) 12 μmol / g, and high brightness with a solid content concentration of 2% by mass containing compound (1) and compound (5) was performed. A fluorescent latex dispersion was prepared.

The same operation was performed for latex particles having other average particle diameters (303 nm, 220 nm, 129 nm, 100 nm, 72 nm), and the same operation was performed for compound (1) only, compound (5) only, and compound (1) and compound (5). A high-intensity fluorescent latex dispersion having a solid content concentration of 2% by mass was prepared.

<5-3> Preparation of high-intensity fluorescent latex particles modified with anti-progesterone antibody
The same operation as in <3> was carried out using each of the high-intensity fluorescent latex particles having a solid content concentration of 2% by mass prepared in <5-2>, and the average particle diameters were 303 nm, 220 nm, 151 nm, 129 nm, 100 nm, and 72 nm. A 1% by mass solution of anti-progesterone antibody-bound high-intensity fluorescent latex particles was prepared.

<6> Preparation of Fluorescent Labeled Particles and Dry Particles of Non-Fluorescent Labeled Particles 280 μL of ultra-pure water, 427 μL of 12.5 mass% sucrose aqueous solution, 133 μL of 20 mass% BSA aqueous solution, 1 mass% anti-progesterone antibody-modified fluorescent latex particles ( 80 μL of 1% by mass anti-T4 antibody modified latex particles (average particle size 150 nm) (80 μL) were mixed. A cup based on polypropylene (Prime Polypro Random PP Grade, manufactured by Prime Polymer Co., Ltd.) was prepared and spotted at 15 μL. Then, using a super dry dryer (TOYO Living Co., Ltd., Ultra Super Dry 00 series), the particles were dried over 12 hours until the water content became 25% by mass or less to prepare dried particles. For the dry particles used for other experimental levels, the average particle size of the latex particles and the mass ratio of the anti-progesterone antibody-modified fluorescent latex particles to the anti-T4 antibody-modified latex particles were appropriately changed as shown in Table 3. To prepare dry particles.

<7> Preparation of substrate <7-1> Preparation of solution of citrate buffer solution of progesterone-BSA conjugate 150 μg of progesterone-BSA conjugate (manufactured by BIO-RAD), 1 mL of citrate buffer solution having a concentration of 50 mmol / L It was added to (pH 5.2, 150 mmol / L NaCl) and dissolved to obtain a solution of citrate buffer.

<7-2> Preparation of anti-mouse antibody A mouse-derived globulin (LAMPIRE Biologic Laboratories, Catalog No. 7404302, Mouse Gamma Globulin Salt Fractionation, 500 mg) was prepared, and an emulsion mixed with a complete Freund's adjuvant (CFA) was administered for the first time. Then, for the 2nd to 4th immunization, an emulsion mixed with an incomplete adjuvant (IFA) was administered, and the goat was immunized with immunosensitization (subcutaneous immunization) four times at 2-week intervals. Then, after confirming an increase in antibody titer by ELISA measurement, whole blood was collected and antiserum was obtained by centrifugation. Then, it was purified by a Protein A column (Pierce Protein A Colons manufactured by Thermo Scientific, Catalog No. 20356) to obtain the desired anti-mouse antibody.

<7-3> Preparation of Progesterone-BSA Combined Fixing Substrate A polymethylmethacrylate (PMMA) substrate (Acrypet VH manufactured by Mitsubishi Rayon Co., Ltd.) was prepared, and the detection region and the reference region were separated by the magnetron sputtering method. A gold film having a thickness of 45 nm was prepared at two locations on one side so as to have a width of 4 mm and a length of 3 mm, respectively, to prepare chips for forming a substrate. A substrate on which the progesterone-BSA conjugate was immobilized by instilling a solution of the progesterone-BSA conjugate prepared in <7-1> with a citrate buffer solution on the gold film surface of the detection region of this chip and drying the solution. Was produced. In addition, a solution containing the anti-mouse antibody prepared in <7-2> (concentration: 50 μg / mL in 50 mmol / L MES buffer pH 6,150 mmol / L NaCl) was instilled in the reference region of each substrate. , Dried.

<8> Cleaning and blocking of the substrate Before attaching the substrate prepared in this manner to the flow path of the sensor chip, a cleaning solution prepared in advance (0.05 mass% Tween® 20 (polyoxyethylene (20)). A PBS solution (pH 7.4) containing sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.) was washed repeatedly 3 times with 300 μL. After washing, 300 μL of PBS solution (pH 7.4) containing 1% by mass casein (manufactured by Thermo Scientific) was added to block the unadsorbed portion of the antibody on the gold thin film, and the mixture was allowed to stand at room temperature for 1 hour. bottom. After washing with the above washing solution, 300 μL of Immunoassay Stabilizer (manufactured by ABI) was added as a stabilizer, left at room temperature for 30 minutes, the solution was removed, and water was completely removed using a dryer.

<9> Manufacture of sensor chip
The prepared substrate was enclosed in a flow path to prepare a flow path type sensor chip so as to have the configuration of the second embodiment of Japanese Patent Application Laid-Open No. 2010-190880. The schematic view is shown in FIGS. 1 and 2. FIG. 1 is a schematic view of the sensor chip 1, and FIG. 2 is an exploded view of the sensor chip 1. The sensor chip 1 is composed of an upper member 2, an intermediate member 3, and a substrate 4. The upper member 2 is provided with a first container 5 and a second container 6. The first container 5 and the second container 6 are collectively referred to as a container group 7. A flow path 10 is formed on the substrate 4, and a detection region 8 and a reference region 9 are formed on the flow path 10.

<10> Preparation of test sample Various concentrations (0.00 ng / mL, 0.5 ng / mL, 2.0 ng / mL, 15.0 ng / mL, 30.0 ng / mL, 45.0 ng) for calibration curve evaluation A sample containing progesterone (/ mL) was prepared.
In addition, for performance evaluation, oriental beagle dog serum purchased from Kitayama Labes was used as dog serum, and test samples (samples) Nos. 1 to 11 were prepared.

<11> Immunoassay of progesterone using fluorescent particles
The dry particles in the cup prepared in <6> are stored in an environment of 25 ° C. and 50% RH (relative humidity) for 15 days, and then the cup contains various concentrations of progesterone prepared in <10>. A mixed sample prepared by sufficiently mixing 100 μL of each of the sample and the test sample (sample) and 44 μmol of magnesium chloride was instilled and mixed with stirring for 10 minutes to obtain a mixed solution. Next, the obtained mixed liquid was instilled on the flow path type sensor chip in which the substrate was sealed, which was produced in <9>. After the instillation, the mixed solution 1 was allowed to flow down at a rate of 10 μL / min while sucking with a pump. The fluorescence intensity on the surface of the gold thin film on which the progesterone-BSA conjugate was fixed was continuously measured for 1.5 minutes. The rate of increase of the obtained fluorescence intensity in a unit time was determined as the fluorescence signal value.

<12> Preparation of calibration curve It is described in the document "The Immunoassay Handbook Third Edition Edition by David Wild (2005)" that a 4-parameter logistic curve model of a sigmoid function can be applied as a calibration curve of a competitive method. , Various samples containing progesterone measured in <11> for each of the comparative examples and examples shown in Table 3 below, using the least squares method generally known as a method for obtaining an approximate curve. Passes near each point of fluorescence signal value at concentration (0.00 ng / mL, 0.5 ng / mL, 2.0 ng / mL, 15.0 ng / mL, 30.0 ng / mL, 45.0 ng / mL) A 4-parameter logistic curve was obtained and used as a calibration curve.

From the calibration curve obtained as described above, the test sample (sample) No. The measured values of each progesterone concentration of 1 to 11 were calculated.

Performance was judged by whether or not it met the standard of the calibration curve. The standard for the calibration curve was determined at two points. The first is the slope of the calibration curve in the low concentration range of progesterone, and the reciprocal was taken and the standard was set to be within 1.4. The second is the deviation (deviation) of the measurement point in the high concentration range of progesterone from the calibration curve, and the standard is within 2%. Within the range of these standards, the coefficient of variation of the measured value can be achieved within 10%, and the accuracy can be achieved within 10%. Highly accurate measurement is possible.

For the concentration of progesterone in the low concentration range determined as a standard, the slope of the calibration curve at 0.5 ng / mL, which is the minimum concentration of clinically significant progesterone, was determined. In addition, for the high concentration range determined as a standard, the deviation (deviation) from the calibration curves of the progesterone concentration of 30.0 ng / mL and 45.0 ng / mL was calculated, and the average value was calculated and evaluated. The results are summarized in Table 3.

<13> Measurement by control machine Measurement of test substance in test sample by Siemens IMMULYZE1000 fully automatic immunochemiluminescence measuring device, which is a large machine widely used by those skilled in the art, according to the instruction manual. Was done. The measurement was performed on the test samples (samples) Nos. 1 to 11 prepared in <10>. The present invention is an invention that enables quick and easy measurement based on the measured value measured by the control machine, and it is necessary that the difference from the measured value of the control machine is small, and the test sample (specimen). For No. 1, the measured value of progesterone obtained from a large machine and 12. The progesterone measurement value (progesterone measurement value of the present invention) obtained from the calibration curve prepared in 1 was evaluated according to the following criteria, and the results are shown in Table 3.

Calculation formula of the deviation width (%) from the large machine

<Evaluation criteria>
For the reciprocal of the calibration curve slope in the low concentration range, the judgment when it was within 1.4 was defined as A, the judgment when it was greater than 1.5 was defined as B, and the determination of A was defined as the allowable range.
Regarding the deviation from the calibration curve in the high concentration range, the judgment when it is less than 1.5% is A, the judgment when it is 1.5% or more and 2% or less is B, and the judgment when it is larger than 2% is C. The judgments of A and B were set as the allowable range.
The deviation width (%) from the large machine is A and B, where the judgment of less than 2.0% is A, the judgment of 2.0% or more and less than 5.0% is B, and the judgment of 5.0% or more is C. Was set as the permissible range.

<14> Measurement of particle fluorescence intensity (relative value) The fluorescence latex dispersion having a solid content concentration of 2% by mass was diluted 200-fold with ultrapure water, and the fluorescence spectrophotometer RF-5300PC (manufactured by Shimadzu Corporation) was excited. The light was set to 658 nm and the measurement was performed. When the fluorescence intensity of the fluorescent latex dispersion was so high that it exceeded the measurement range, it was diluted with ultrapure water until the maximum value of the fluorescence intensity was measurable. The integral value of the fluorescence intensity of the emission spectrum of the fluorescent latex dispersion with respect to the integral value of the fluorescence intensity of the emission spectrum of the fluorescence latex dispersion prepared in <6> was defined as the particle fluorescence intensity (relative value). The calculation formula used for the calculation is shown below.
Fluorescence intensity (relative value) = (integral value of fluorescence intensity of emission spectrum of fluorescent latex dispersion) / (integral value of fluorescence intensity of emission spectrum of fluorescence latex dispersion prepared in <6>)

The results are shown in Table 3.

From the results in Table 3, it was found that by using the high-luminance particles of the present invention and the unlabeled particles, it was possible to measure with high accuracy over the entire measurement range, and the effect of the present invention was confirmed.

1 Sensor chip 2 Upper member 3 Intermediate member 4 Substrate 5 First container 6 Second container 7 Container group 8 Detection area 9 Reference area 10 Flow path

Claims (20)

A labeled first particle modified with a first binding agent that has specific binding to progesterone,
An unlabeled second particle modified with an antibody against an antigen other than progesterone,
A flow path for flowing the first particle and the second particle, and
A substrate having a substance having a binding property to the first binding substance,
A progesterone measurement kit that contains
The first particle having the above-mentioned label contains at least one compound represented by the following formula (1) and the particle.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. Large particle size,
kit.

In the formula, R ^{11 to} R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group. , Or arylthio groups, which may have substituents, and ^{at least three of R 11 to} R ¹⁵ represent atoms or groups other than hydrogen atoms. X ¹ and X ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group. It may have a substituent and X ¹ and X ² may be linked to each other to form a ring. Ar ¹ and Ar ² each independently represent an aryl group or a heterocyclic group, which may have a substituent. L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-. The kit according to claim 1, wherein the first particle and the second particle are latex particles. The first particle having the label is a luminescent particle containing at least one energy donor compound, at least one energy acceptor compound, and particles, and the energy donor compound and the energy acceptor compound. The kit according to claim 1 or 2, wherein at least one of the above is a compound represented by the formula (1). The third aspect of the present invention, wherein the energy donor compound contains at least one compound represented by the formula (1), and the energy acceptor compound contains at least one compound represented by the formula (1). Kit. The kit according to claim 3 or 4, wherein the molar ratio of the energy donor compound to the energy acceptor compound is 1:10 to 10: 1. The kit according to any one of claims 1 to 5, wherein the mass ratio of the second particle to the first particle is 1 to 20. The kit according to any one of claims 1 to 6, wherein the first binding substance having specific binding property to progesterone is an antibody. (I) (a) Labeled first particles modified with a first binding agent having a specific binding property to progesterone.
(B) An unlabeled second particle modified with an antibody against an antigen other than progesterone.
(C) A test sample containing progesterone and
To obtain a mixture by mixing
(Ii) A step of applying the mixture obtained in the above step (i) onto a substrate, and
(Iii) A step of capturing the first binding substance at a reaction site on the substrate having a substance having a binding property to the first binding substance.
(Iv) A step of detecting a labeled first particle modified with the first binding agent captured on the reaction site.
A method for measuring progesterone, including
The first particle having the above-mentioned label contains at least one compound represented by the following formula (1) and the particle.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. Large particle size,
How to measure progesterone.

In the formula, R ^{11 to} R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group. , Or arylthio groups, which may have substituents, and ^{at least three of R 11 to} R ¹⁵ represent atoms or groups other than hydrogen atoms. X ¹ and X ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group. It may have a substituent and X ¹ and X ² may be linked to each other to form a ring. Ar ¹ and Ar ² each independently represent an aryl group or a heterocyclic group, which may have a substituent. L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-. The method according to claim 8, wherein the first particle and the second particle are latex particles. The first particle having the label is a luminescent particle containing at least one energy donor compound, at least one energy acceptor compound, and particles, and the energy donor compound and the energy acceptor compound. The method according to claim 8 or 9, wherein at least one of the above is a compound represented by the formula (1). The tenth aspect of the present invention, wherein the energy donor compound contains at least one compound represented by the formula (1), and the energy acceptor compound contains at least one compound represented by the formula (1). the method of. The method according to any one of claims 8 to 11, wherein the mass ratio of the second particle to the first particle is 1 to 20. Any one of claims 8 to 12, wherein in step (iv), the first labeled particle modified with the first binding material captured on the reaction site is detected by surface plasmon fluorescence. The method described in the section. (A) a labeled first particle modified with a first binding agent that has specific binding to progesterone, and (b) an unlabeled first particle modified with an antibody against an antigen other than progesterone. A progesterone measurement reagent containing two particles.
The first particle having the above-mentioned label contains at least one compound represented by the following formula (1) and the particle.
The average particle size of the first particle is 50 to 250 nm, the average particle size of the second particle is 70 to 500 nm, and the average particle size of the second particle is larger than the average particle size of the first particle. Large particle size,
Progesterone measurement reagent.

In the formula, R ^{11 to} R ¹⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group. , Or arylthio groups, which may have substituents, and ^{at least three of R 11 to} R ¹⁵ represent atoms or groups other than hydrogen atoms. X ¹ and X ² independently represent a halogen atom, an alkyl group, an aryl group, a heterocyclic group, a hydroxy group, an alkoxy group, an aryloxy group, an alkylthio group, an arylthio group, an ethenyl group, or an ethynyl group. It may have a substituent and X ¹ and X ² may be linked to each other to form a ring. Ar ¹ and Ar ² each independently represent an aryl group or a heterocyclic group, which may have a substituent. L ¹ and L ² independently represent any of the formulas (L-1) to (L-4).

In the formula, R ^{111 to} R ¹¹⁶ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group and alkylthio group, respectively. , Or arylthio groups, which may have substituents. A represents -O-, -S-, or -NH-. The progesterone measuring reagent according to claim 14, wherein the compound represented by the formula (1) is a compound represented by the following formula (3).

In the formula, R ¹¹ , R ¹² , R ¹⁴ , R ¹⁵ , X ¹ , X ² , Ar ¹ , Ar ² , L ¹ and L ² are synonymous with the definitions in formula (1), except that R ¹¹ , At ^{least two of R 12} , R ¹⁴ and R ¹⁵ are atoms or groups other than hydrogen atoms. R ^{31 to} R ³⁵ are independently hydrogen atom, halogen atom, alkyl group, aryl group, heterocyclic group, ethenyl group, ethynyl group, amino group, acyl group, alkoxy group, aryloxy group, alkylthio group, or arylthio. Representing a group, these may have a substituent ^{, and any one of R 31} , R ³² , R ³⁴ and R ³⁵ is a group consisting of two or more atoms. The progesterone measuring reagent according to claim 14, wherein the compound represented by the formula (1) is a compound represented by the following formula (4).

In the formula, R ¹² , R ¹³ , R ¹⁴ , X ¹ , X ² , Ar ¹ , Ar ² , L ¹ and L ² are synonymous with the definitions in formula (1), except that R ¹² , R ¹³ and At ^{least one of R 14} is an atom or group other than a hydrogen atom. R ⁴¹ and R ⁴² each independently represent an aryl group, a heterocyclic group, an ethenyl group, or an ethynyl group, which may have a substituent. The progesterone measuring reagent according to claim 14, wherein the compound represented by the formula (1) is a compound represented by the following formula (5).

In the formula, R ^{11 to} R ¹⁵ , X ¹ , X ² , L ¹ and L ² are synonymous with the definition in the formula (1). R ⁵¹ and R ⁵² each independently represent an alkyl group, an aryl group, a heteroaryl group, an amino group, an acyl group, an alkoxy group, an aryloxy group, an alkylthio group, or an arylthio group, and these have a substituent. You may. Q ¹ and Q ² each independently represent an aromatic hydrocarbon ring or aromatic hetero ring, which may have a substituent. The progesterone measuring reagent according to any one of claims 14 to 17, wherein the mass ratio of the second particle to the first particle is 1 to 20. The progesterone measuring reagent according to any one of claims 14 to 18, wherein the first binding substance having specific binding property to the progesterone is an antibody. The progesterone measuring reagent according to any one of claims 14 to 19, wherein the first particle having the label is a fluorescent latex particle and the second particle is a latex particle.

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